JP2011526243A - Immune-based botulinum toxin serum type A activity assay - Google Patents

Abstract

Herein SNAP-25 composition, the alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product how to create, SNAP-25 α-SNAP- 25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / a cleavage site scissile bond from, detects the BoNT / a activity And a method for detecting an α-BoNT / A neutralizing antibody.

Description

  This patent application claims priority to US Provisional Patent Application No. 61/036723, filed March 14, 2008, in accordance with 35 U.S.C. §119 (e), which is incorporated by reference in its entirety. As a part of this specification.

  Clostridium toxins such as botulinum neurotoxin (BoNT), BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F and BoNT / G and tetanus neurotoxin (TeNT) are transmitted through neurons. The ability to block is utilized in a variety of therapeutic and cosmetic applications, see, for example, William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004). Clostridial toxins that are commercially available as pharmaceutical compositions include, for example, BOTOX® (Allergan, Inc., Irvine, Calif.), DYSPORT® / RELOXIN®, (Ipsen Ltd., Slough, England), PURTOX® (Mentor Corp., Santa Barbara, CA), XEOMIN® (Merz Pharmaceuticals, GmbH., Frankfurt, Germany), NEURONOX® (Medy-Tox, Inc., Ochang -Myeon, South Korea), BoNT / A preparations such as BTX-A (Biogen-tech Ltd., University, Yantai, Shandong, China); and for example MYOBLOC® / NEUROBLOC® (Solstice Neurosciences BoNT / B preparations such as, Inc., South San Francisco, Calif.). Illustratively, BOTOX® is currently used in the United States to treat adults with spastic torticollis to reduce the severity of head position abnormalities and neck pain associated with spastic torticollis; Approved for treatment of severe primary axillary hyperhidrosis that is not done; and for treatment of strabismus and blepharospasm associated with dystonia, including benign essential blepharospasm or VII nerve injury in patients over 12 years of age.

The murine LD ₅₀ bioassay, currently a lethal test, is still the “gold standard” used by all pharmaceutical manufacturers to express the efficacy of the preparation. SS Arnon et al., JAMA 285: 1059-1070 (2001). In fact, the label unit of the pharmaceutical formulation is mouse LD ₅₀ units, and the number of animals required to generate statistically useful LD ₅₀ data is large. The advantage of the mouse LD ₅₀ bioassay is that all the steps required for botulinum toxin uptake are taken, instead of determining activity for only part of this addiction process, such as an in vitro assay that measures only light chain enzyme activity. (Eg toxin binding to cell surface receptors, internalization of toxin-receptor complexes, light chain translocation to the cytoplasm, light chain cleavage of the substrate). Unfortunately, the mouse LD ₅₀ bioassay has a high operating cost due to the large number of laboratory animals required, the lack of specificity because all BoNT serotypes will cause the same measurable endpoint, and It suffers from a number of drawbacks, including the potential for inaccuracy when not using large groups of animals. In addition, animal protection groups have reduced animal testing and, more importantly, oversight of the US (FDA / NICEEATM / ICCVAM) and Europe (MHRA and EDQM) to replace the mouse LD ₅₀ bioassay for product release It puts pressure on government agencies and pharmaceutical companies that produce botulinum neurotoxin products. Regulatory agencies have promised pharmaceutical companies to apply the three “R” principles for botulinum neurotoxin efficacy testing: Reduce, Refine, Replace. “Advances in the application of the three Rs to the efficacy test of botulinum toxin type A” D. Straughan, Altern. Lab. Anim. 34 (3): 305-313 (2006). In recent years, several steps have already been employed to reduce and refine the mouse LD ₅₀ bioassay to standardize the protocol and generate more consistent data using fewer animals per assay.

  Therefore, such non-animal based assays alleviate the need for animal testing and all the disadvantages, costs and ethical issues associated with this type of animal based assay, so botulinum toxin A simple, reliable, validated, and government-acceptable botulinum toxin activity assay that can assess the integrity of all steps required for uptake would be of significant value . The present description provides novel compositions, cells and methods for assaying the activity of botulinum toxin A useful in various industries such as, for example, the pharmaceutical and food industries, and provides related advantages as well. . Such compositions, cells and methods do not use living animals or tissues taken from living animals, but can evaluate all the steps necessary for neurotoxic effects.

FIG. 1 shows a schematic of the current paradigm of neurotransmitter release and clostridial toxin intoxication in central and peripheral neurons. FIG. 1A shows a schematic diagram of the neurotransmitter release mechanism of central and peripheral neurons. The release process can be described as comprising two steps: 1) vesicle docking, where vesicle-bound SNARE proteins containing neurotransmitter molecules associate with membrane-bound SNARE proteins located in the cell membrane And 2) neurotransmitter release, where the vesicles fuse with the cell membrane and neurotransmitter molecules are excreted by exocytosis. FIG. 1B shows a schematic diagram of the toxic mechanism of tetanus and botulinum toxin activity in central and peripheral neurons. This poisoning process can be described as comprising four steps: 1) receptor binding, where the clostridial toxin binds to the clostridial receptor complex and initiates the poisoning process; 2) the complex After internalization, where toxin binding, vesicles containing the toxin / receptor complex are taken up by the cell by endocytosis; 3) light chain translocation, where the vesicle's internal pH changes, clostridial toxin weight formation of a channel pore comprising the H _N domain of a chain, separation of the light chain from a Clostridial toxin heavy chain, and a plurality of considered event occurs, including the release of the light chain; and 4) the target modification by the enzyme, wherein Clostridial toxin light chain is proteolyzed by its target SNARE substrate such as SNAP-25, VAMP or syntaxin. Cleave, thereby preventing vesicle docking and neurotransmitter release. FIG. 2 shows a comparison of BoNT / A uptake in four cell lines by Western blot analysis. FIG. 2A shows a graph of SNAP-25 cleavage products detected based on the amount of BoNT / A used to treat the cell line. Data was analyzed with SigmaPlot using a four parameter logistic model and EC ₅₀ values were obtained for each cell line. The ranking of detected SNAP-25 cleavage product signals was: SiMa >>Neuro-2a>LA1-55n> PC12. FIG. 2B shows that the signal to noise ratio of the raw signal at 300 pM to 0 pM and 1.2 pM to 0 pM was calculated for the assay. SiMa cells yielded the highest signal to noise ratio and the lowest _{The EC 50} values. FIG. 3 shows the optimization of cell differentiation media for established cell lines useful in the immuno-based methods of detecting BoNT / A activity disclosed herein. FIG. 4 shows optimization of cell differentiation time for cells comprising established cell lines useful in the immuno-based methods of detecting BoNT / A activity disclosed herein. FIG. 5 shows the optimization of BoNT / A treatment of cells comprising established cell lines useful in the immuno-based methods of detecting BoNT / A activity disclosed herein. The results indicate that an EC _{50 of} less than 2 pM was achieved with any BoNT / A treatment tested. FIG. 6 shows the sensitivity of the immune-based method for detecting BoNT / A activity disclosed herein. The results indicate that cellular uptake of BoNT / A took less than 1 minute before producing significant amounts of SNAP-25 cleavage product above background. FIG. 7 shows the specificity of the immune-based method for detecting BoNT / A activity disclosed herein. The results show that the immune-based methods for detecting BoNT / A activity disclosed herein can measure all steps involved in BoNT / A intoxication. FIG. 8 shows a dose response curve of differentiated SiMa cells treated with the BoNT / A complex using the immuno-based method of detecting BoNT / A activity disclosed herein. FIG. 9 shows the results of an immuno-based BoNT / A activity assay for formulating BoNT / A pharmaceuticals using the immuno-based method of detecting BoNT / A activity disclosed herein. FIG. 10 shows the detection of α-BoNT / A neutralizing antibodies in human serum using the immuno-based method of detecting BoNT / A activity disclosed herein.

[Detailed description]
The present specification provides a novel assay for determining the presence or absence of active BoNT / A in a sample and for determining the activity / efficacy of a BoNT / A preparation. The novel cell-based assays disclosed herein rely on cells, reagents and detection methods that allow the assay to detect picomolar concentrations of BoNT / A in a sample. The cell-based assays disclosed herein reduce the need for animal toxicity studies, yet analyze multifunctional BoNT / A, ie, toxin binding and cellular uptake, translocation to the cell cytosol, and protease activity To help. As discussed further below, the novel methods and compositions can be used to analyze crude and bulk samples and highly purified double-chain toxins and formulated toxin products, and further automated high-throughput assays Easy to handle in form.

Thus one of the embodiments disclosed herein may bind an epitope comprising a carboxyl-terminus at the P ₁ residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product α- Compositions for producing SNAP-25 antibodies are provided. The composition can comprise a composition comprising an adjuvant and a SNAP-25 antigen, a carrier linked to a SNAP-25 antigen or a carrier linked to a mobile spacer linked to a SNAP-25 antigen, wherein A mobile linker is interposed between the SNAP-25 antigen and the carrier. Eliciting an immune response that generates the SNAP-25 α-SNAP-25 antibodies that can bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from Any and all SNAP-25 antigens include, but are not limited to, SNAP-25 antigens derived from naturally occurring SNAP-25, SNAP-25 antigens derived from non-naturally occurring SNAP-25, and SNAP- It is envisioned that it may be useful as a SNAP-25 antigen, including a SNAP-25 antigen comprising an immunoreactive fragment of SNAP-25 from 25, a naturally occurring SNAP-25 or a non-naturally occurring SNAP-25. SNAP-25 BoNT / A cleavage site scissile in _{the P 1} residue from the binding can bind an epitope comprising a carboxyl-terminus alpha-SNAP-25 useful SNAP for generating antibodies from cleavage product SNAP-25 comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a carrier peptide comprising, but not limited to, SEQ ID NO: 38 Antigen is included. SNAP-25 cleavage BoNT from the product / A cleavage site scissile in _{the P 1} residue from the binding can bind an epitope comprising a carboxyl-terminus alpha-SNAP-25 Other useful to generate antibodies The composition includes, but is not limited to, a composition comprising a carrier linked to a mobile linker linked to a SNAP-25 antigen having a carboxylated C-terminal glutamine, wherein the mobile The sex linker is interposed between the SNAP-25 antigen and the carrier. Any and all adjuvants including but not limited to polyethylene glycol (PEG), monomethoxy polyethylene glycol (mPEG), polyvinyl alcohol (PVA), Freund's complete and incomplete adjuvants may be useful in such compositions. It is assumed that

Another aspect disclosed herein is α- capable of binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. Methods for producing SNAP-25 antibodies are provided. Embodiments of this method include: (a) administering a composition disclosed herein to an animal; (b) collecting a sample containing α-SNAP-25 antibody or α-SNAP-25 antibody-producing cells from the animal And (c) isolating the α-SNAP-25 antibody from the sample. The disclosed methods include an α-SNAP-25 monoclonal antibody capable of binding to an epitope comprising a carboxyl terminal glutamine from a BoNT / A cleavage site cleavable linkage from a SNAP-25 cleavage product, or a SNAP-25 cleavage It is useful for making any of the α-SNAP-25 polyclonal antibodies that can bind to an epitope comprising a carboxyl-terminal glutamine from a BoNT / A cleavage site cleavable linkage from the product.

Yet another aspect disclosed herein is α- capable of binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. SNAP-25 antibodies are provided. Such α-SNAP-25 antibodies include both naturally occurring and non-naturally occurring antibodies, as well as monoclonal α-SNAP-25 or polyclonal α-SNAP-25 antibodies. The SNAP-25 cleavage BoNT / A cleavage site scissile _{P 1} residue with useful as alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus monoclonal alpha-SNAP-25 antibody from binding from the product Include, but are not limited to, monoclonal α-SNAP-25 antibodies generated from hybridoma cell lines 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2.

Yet another aspect disclosed herein provides a method of detecting BoNT / A activity. Embodiments of this method include (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line are sensitive to BoNT / A intoxication; (b) that the treated SNAP-25 comprising the cleavage product SNAP-25 component having a carboxyl-terminus at _{the P 1} residue from cells BoNT / a cleavage site scissile bond is isolated; (c) An α-SNAP-25 antibody capable of binding a SNAP-25 component to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product; And (d) detecting the presence of an antibody-antigen complex comprising α-SNAP-25 antibody and SNAP-25 cleavage product; wherein antibody-anti Detection by complex is indicative of BoNT / A activity; comprising the steps. The α-SNAP-25 antibody of step c can optionally be linked to a solid support.

Yet another aspect disclosed herein provides a method of detecting BoNT / A activity. Embodiments of this method include: (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line can take up BoNT / A; (b) treated with SNAP-25 component comprising a SNAP-25 having a carboxyl-terminus from cells in _{the P 1} residue of the BoNT / a cleavage site scissile bond can be isolated; (c) SNAP-25 Contacting the component with an α-SNAP-25 antibody capable of binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. And (d) detecting the presence of an antibody-antigen complex comprising an α-SNAP-25 antibody and a SNAP-25 cleavage product; wherein the antibody-antigen complex Detection is indicative of BoNT / A activity by; comprising the steps. The α-SNAP-25 antibody of step c can optionally be linked to a solid support.

A further aspect disclosed herein provides a method for determining BoNT / A immune resistance in a mammal. Embodiments of this method include (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibody; (b) from an established cell line The cells from the established cell line are sensitive to BoNT / A intoxication; (c) the P ₁ residue of the BoNT / A cleavage site cleavable binding from the treated cells. Isolating a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl terminus at the group; (d) converting the SNAP-25 component into a BoNT / A cleavage site from the SNAP-25 cleavage product. contacting a _{P 1} residues may be attached to an epitope comprising a carboxyl-terminus α-SNAP-25 antibody from scissile bond; (e) α-SNAP- 25 antibody Contact And detecting the presence of an antibody-antigen complex comprising a SNAP-25 cleavage product; (f) repeating steps ae with a negative control sample instead of the test sample; and (g) step (e The amount of antibody-antigen complex detected in step (f) is compared with the amount of antibody-antigen complex detected in step (f), wherein the amount of antibody-antigen complex detected in step (f) The lower detected amount of antibody-antigen complex detected in step (e) is an indicator of the presence of α-BoNT / A neutralizing antibody. The α-SNAP-25 antibody of step d may optionally be linked to a solid support. The control sample in step f can also include a positive control sample in addition to the negative control sample.

  Clostridium toxin produced by Clostridium botulinum, Clostridium tetani, Clostridium baratii and Clostridium butyricum is a therapeutic and cosmetic treatment in humans and other mammals. It is the most widely used. The strain of Clostridium botulinum produces seven antigenically distinct botulinum toxin serotypes (BoNT), which are human (BoNT / A, BoNT / B, BoNT / E and BoNT / F), animals (BoNT / C1) And BoNT / D) or those isolated from soil (BoNT / G) have been identified by investigating botulism. All seven botulinum toxin serotypes have similar structural and biological properties, but each also displays heterogeneous features such as different pharmacological properties. In contrast, tetanus toxin (TeNT) is produced by a homogeneous group of tetanus bacteria. Two other species of Clostridium, Clostridium balati and Clostridium butyricum, also produce toxins similar to BoNT / F and BoNT / E, respectively.

Clostridial toxins are each translated as a single-chain polypeptide of approximately 150 kD, which is subsequently cleaved by proteolytic cleavage within the disulfide loop by naturally occurring proteases such as endogenous clostridial toxin proteases or naturally occurring proteases produced in the environment. Is done. This post-translational processing results in a double chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) that are maintained together by a single disulfide bond and non-covalent interactions. . Each mature double-chain molecule is located in an LC containing three functionally distinct domains: 1) a metalloprotease region containing a zinc-dependent endopeptidase activity that specifically targets the core components of the neurotransmitter release device 2) a translocation domain contained within the amino-terminal half of HC (H _N ) that promotes the release of LC from intracellular vesicles into the cytoplasm of the target cell; and 3) the surface of the target cell A binding domain found in the carboxyl-terminal half of HC (H _C ) that determines the binding activity and binding specificity of the toxin to the receptor complex located in

  The binding, translocation and enzyme activity of the three functional domains are all necessary for toxicity. Although all details of this process are still not accurately understood, the overall cell poisoning mechanism by which clostridial toxin enters neurons and blocks neurotransmitter release is similar regardless of serotype or subtype. Applicant will never want to be limited by the following description, but the addiction mechanism can be described as comprising at least four steps: 1) receptor binding; 2) complex internalization; 3 ) Light chain translocation; and 4) target modification by the enzyme (FIG. 1). The process is initiated when the HC domain of a clostridial toxin binds to a toxin-specific receptor system located on the cell membrane surface of the target cell. The binding specificity of the receptor complex is believed to be achieved in part by the specific combination of ganglioside and protein receptors that would clearly comprise a clostridial toxin receptor complex. Once bound, the toxin / receptor complex is internalized by endocytosis and the internalized vesicles are classified into specific intracellular pathways. The translocation process appears to be triggered by acidification of the vesicular compartment. This process appears to initiate a critical pH-dependent structural rearrangement that increases hydrophobicity, promotes pore formation, and promotes separation of toxin heavy and light chains. Once separated, the toxin light chain endopeptidase is released from intracellular vesicles into the cytosol, where it appears to specifically target the core components of the neurotransmitter release device. These core proteins, vesicle associated membrane protein (VAMP) / synaptobrevin, 25 kDa synaptosome associated protein (SNAP-25) and syntaxin are required for synaptic vesicle docking and nerve terminal fusion, and soluble N-ethylmaleimide It constitutes a member of the sensitivity factor attachment protein receptor (SNARE) family. BoNT / A and BoNT / E cleave SNAP-25 at the carboxyl terminal region to release 9 or 26 amino acid fragments, respectively, and BoNT / C1 also cleaves SNAP-25 near the carboxyl terminus. Eight amino acid fragments are released. Botulinum serotypes BoNT / B, BoNT / D, BoNT / F and BoNT / G, and tetanus toxin act at the conserved central portion of VAMP and release the amino-terminal portion of VAMP into the cytosol. BoNT / C1 cleaves syntaxin at a single site near the cytoplasmic membrane surface. Selective proteolysis of synaptic SNAREs explains the blockade of neurotransmitter release caused by clostridial toxins in vivo. The SNARE protein target of clostridial toxin is common to various non-neuronal exocytosis; in these cells, light chain peptidase activity, like neurons, blocks exocytosis, eg, “How Botulinum and Tetanus Neurons Does the toxin block neurotransmitter release? "Yann Humeau et al., Biochimie. 82 (5): 427-446 (2000);" Botulinum and tetanus neurotoxins: structure, function and therapeutic utility "Kathryn Turton et al., Trends Biochem. Sci. 27 (11): 552-558 (2002); “The path of tetanus and botulinum neurotoxin in neurons” Giovanna Lalli et al. Trends Microbiol. 11 (9): 431-437 (2003) Please refer to.

Aspects of the disclosure include in part an α-SNAP-25 antibody capable of binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable bond from a SNAP-25 cleavage product The composition for producing | generating this. Other aspects of the present disclosure are in part α-SNAP- capable of binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. An immune response inducing composition for generating 25 antibodies. As used herein, the term “immune response inducing composition” refers to a composition comprising a SNAP-25 antigen, which, when administered to an animal, stimulates an immune response against the SNAP-25 antigen. Thereby generating an α-SNAP-25 antibody capable of binding to an epitope comprising the carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. The term “immune response” refers to any response to an immune response inducing composition by an animal's immune system. Examples of immune responses include, but are not limited to, CTL responses including, for example, antigen-specific induction of CD8 + CTL, helper T cell responses including T cell proliferation response and cytokine release, and B cell responses including, for example, antibody generation responses And local and systemic humoral immunity, such as The term “inducing an immune response” refers to the administration of an immune response inducing composition or polynucleotide encoding an immune response inducing composition, where the immune response is affected, ie stimulated, initiated or elicited.

The composition comprises a SNAP-25 antigen. As used herein, the term “antigen” refers to a molecule that elicits an immune response and includes, but is not limited to, peptides, polysaccharides, and lipid conjugates such as lipoproteins and glycolipids. including. As used herein, the term “SNAP-25 antigen” refers to any antigen having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable bond capable of eliciting an immune response. The SNAP-25 antigen used in the immune response inducing composition must be large enough for the sequence to be substantially unique, thus producing an antibody that is cross-reactive with antigens other than SNAP-25. Reduce the possibility of In addition to elicit only immune response against SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the SNAP-25 antigen used is substantially the BoNT / A cleavage site cleavable bond in the immune response induced composition a must be sufficiently small, hence a carboxyl-terminus at the _{P 1} residue of SNAP-25 and BoNT / a cleavage site scissile bond having a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond There is an increased likelihood of producing α-SNAP-25 antibodies that can be distinguished from the lacking SNAP-25. In addition, with good yields that are reproducible and selective, it binds α-SNAP-25 antibodies of a single amino acid sequence that bind with acceptable avidity to enable the design of highly sensitive assays. It is also highly desirable to create.

Sequences surrounding the BoNT / A cleavage site present in SNAP-25 as _{P 5 -P 4 -P 3 -P 2} -P 1 -P 1 '-P 2' -P 3 '-P 4' -P 5 ' Where P ₁ -P ₁ ′ represents a cleavable bond. During cleavage by BoNT / A, the generated resultant cleavage product fragment and _{P 1 '-P 2' -P 3} '-P 4 containing _{P 5 -P 4 -P 3 -P 2} -P 1 sequence It comprises a fragment containing '-P ₅ '. Thus, as used herein, the term “SNAP-25 having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage” is any term having a P ₁ residue as its carboxyl terminal amino acid. Refers to SNAP-25. For example, Q ₁₉₇ -R ₁₉₈ of human SNAP-25 (SEQ ID NO: 5) represents a P ₁ -P ₁ ′ cleavable bond with respect to the BoNT / A cleavage site. As such, “SNAP-25 with BoNT / A cleavage site cleavable linkage carboxyl-terminal glutamine” is any SNAP-25 cleavage product with glutamine at its carboxyl-terminal amino acid, where glutamine is a cleavable linkage Q represents ₁₉₇ . As another example, Torpedo Marumorata (Torpedo marmorata) SNAP-25 of _K 204 _{-H 205} (SEQ ID NO: 16) represents the _P 1 -P ₁ 'scissile bond about the BoNT / A cleavage site. As such, a “SNAP-25 with a BoNT / A cleavage site cleavable linkage carboxyl-terminal lysine” is any SNAP-25 cleavage product having a lysine at its carboxyl-terminal amino acid, where lysine is a cleavable linkage. K ₂₀₄ is represented.

By modification of the SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond from the BoNT / A cleavage site, immunogenic when not associated SNAP-25 antigen, a hapten or a modified The immunogenicity of any other antigenic compound that is sex, non-immunogenic or weakly immunogenic can be enhanced. In aspects of this embodiment, it is possible to carboxylated carboxyl-terminal P ₁ residue from scissile bond SNAP-25 antigen. Carboxylation increases the desired immunogenic properties of the SNAP-25 antigen in two respects. Since the first charged amino acid enhances immunogenicity, the addition of a COO ^- group to the carboxyl terminal residue will increase the overall immunogenicity of the SNAP-25 antigen. Second, since the P ₁ residue of the BoNT / A cleavage site scissile bond is in a state of being charged at the time of cutting, COO to the carboxyl terminal residue ^- additional groups, alpha-SNAP disclosed herein It would better mimic the actual antigen designed for the -25 antibody to bind.

  In aspects of this embodiment, the SNAP-25 antigen may be, for example, keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachments. The amino terminal residue from the SNAP-25 antigen can be modified by the addition of an amino acid suitable for attachment to a carrier protein such as a multiple attachment peptide (MAP). For example, cysteine residues can be replaced at the amino terminus to conjugate the carrier protein KLH.

In thus embodiment, SNAP-25 antigen, for example, at least 5 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, It may be at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids long. In another embodiment, SNAP-25 antigen, for example at most 5 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond, at most 6, at most 7, at most 8, At most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, It may be at most 19, at most 20, at most 25 or at most 30 amino acids long. In yet another embodiment, BoNT / A SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the cleavage site scissile bond, for example, between 7-12 amino acids, between or 13-18 amino acids 10-15 amino acids Between.

In another embodiment, SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond comprises SEQ ID NO: comprising 32. In aspects of this embodiment, SNAP-25 antigen of SEQ ID NO: having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. In a further embodiment, SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond comprises SEQ ID NO: comprising 38.

In yet another embodiment, SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond comprises SEQ ID NO: comprising 39. In aspects of this embodiment, SNAP-25 antigen of SEQ ID NO: having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. In a further embodiment, SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond comprises SEQ ID NO: comprising 45.

Any and eliciting an immune response that generates the alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product It is envisioned that all SNAP-25 antigens may be useful as SNAP-25 antigens. Therefore, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 The amino acid sequence variant comprising SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148 is a P ₁ residue from the BoNT / A cleavage site cleavable bond from the SNAP-25 cleavage product. It may be useful as a SNAP-25 antigen to elicit an immune response that generates an α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at the group. Thus, in an embodiment, the SNAP-25 antigen comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148 or at least 1, at least 2, at least 3, at least 4 or against a SNAP-25 antigen At least 5 amino acid substitutions, deletions or additions can be substituted. In yet another embodiment, the SNAP-25 antigen comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148 for SNAP-25 antigen comprising at least 70%, at least 75%, It may have at least 80%, at least 85%, at least 90% or at least 95% amino acid identity.

To enhance the immunogenicity of a SNAP-25 antigen that is immunogenic, non-immunogenic or weakly immunogenic in the absence of a carrier, one or more carriers can be linked to the SNAP-25 antigen. That is assumed. Non-limiting examples include, for example, keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptides (MAP). . As is well known in the art, non-antigenic or weakly antigenic antigens can be made antigenic by binding the antigen to a carrier. Various other carriers and methods for binding antigens to carriers are well known in the art. See, for example, Harlow and Lane, supra, 1998a; Harlow and Lane, supra, 1998b; and David W. Waggoner, Jr. et al., “Immunogenicity enhancing carriers and compositions and methods of using the same,” US Patent Application Publication No. See 2004/0057958 (March 25, 2004). An epitope can also be created by expressing the epitope as a fusion protein. Methods for expressing polypeptide fusions are well known to those skilled in the art, for example as described in Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999). . Since the carboxyl end of SNAP-25 antigen must be _{the P 1} residue of the BoNT / A cleavage site scissile bond, a carrier must be linked to the amino end of SNAP-25 antigen.

  In order to enhance the immunogenicity of a SNAP-25 antigen that is immunogenic, non-immunogenic or weakly immunogenic in the absence of a mobile linker, one or more mobile spacers are added to the SNAP-25. It is envisioned that it can be linked to an antigen. The mobile spacer increases the overall peptide length of the SNAP-25 antigen and provides mobility, thereby facilitating proper presentation of the SNAP-25 antigen to immune cells. As a non-limiting example, the composition can comprise a SNAP-25 antigen linked in series to one or more mobile spacers to better present the SNAP-25 antigen to immune cells; Thereby promoting an immune response.

  A mobile spacer comprising a peptide is at least one amino acid long and comprises an uncharged amino acid with a short side chain R group such as glycine, alanine, valine, leucine or serine. Thus, in embodiments, the movable spacer may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids long. In another embodiment, the movable spacer is for example at least 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most It may be 9 or at most 10 amino acids long. In yet another embodiment, the movable spacer may be, for example, between 1-3 amino acids, between 2-4 amino acids, between 3-5 amino acids, between 4-6 amino acids, or between 5-7 amino acids. Non-limiting examples of mobile spacers include G spacers such as GGG, GGGG (SEQ ID NO: 55) and GGGGS (SEQ ID NO: 56) or AAA, AAAA (SEQ ID NO: 57) and AAAAV (SEQ ID NO: 58). A spacer such as The mobile spacer is linked in-frame to the SNAP-25 antigen as a fusion protein.

As discussed above, mobile spacers are used in part to increase the overall peptide length of the SNAP-25 antigen. For example, a 5-10 amino acid SNAP-25 antigen may have its overall length increased by linking a 3-5 amino acid mobile spacer to the amino terminus of the SNAP-25 antigen. As another example, a 5-10 amino acid SNAP-25 antigen may have its overall length increased by linking a 4-6 amino acid mobile spacer to the amino terminus of the SNAP-25 antigen. As another example, a 5-10 amino acid SNAP-25 antigen may have its overall length increased by linking a 7-10 amino acid mobile spacer to the amino terminus of the SNAP-25 antigen. As another example, a 7-12 amino acid SNAP-25 antigen may have its overall length increased by linking a 1-3 amino acid mobile spacer to the amino terminus of the SNAP-25 antigen. As another example, a 7-12 amino acid SNAP-25 antigen may have its overall length increased by linking a 4-6 amino acid mobile spacer to the amino terminus of the SNAP-25 antigen. The increased length provided by the mobile spacer allows for the selection of small size SNAP-25 antigens, whereby the SNAP-25 antigen is substantially the P ₁ residue of the BoNT / A cleavage site cleavable bond. in expected only to elicit an immune response against the SNAP-25 having a carboxyl-terminus increases, thus the BoNT / a cleavage site scissile bond _{the P 1} residue with the BoNT / a cleavage site SNAP-25 having a carboxyl-terminus There is an increased likelihood of generating α-SNAP-25 antibodies that can be distinguished from SNAP-25 lacking the carboxyl terminus at the P ₁ residue of the cleavable linkage.

It is envisioned that the compositions disclosed herein can optionally comprise a SNAP-25 antigen disclosed herein and one or more adjuvants. As used herein, the term “adjuvant” when used with reference to a SNAP-25 composition refers to any substance or substance that increases or diversifies the immune response to a SNAP-25 antigen. Refers to a mixture. Adjuvants are useful, for example, to reduce the number of immunizations or the amount of antigen required for protective immunity. The use of adjuvants in immune response inducing compositions is well known. The main purpose of these adjuvants is to allow an increased immune response. Non-limiting adjuvants include, for example, liposomes, but are not limited to, for example, Freund's complete adjuvant (FCA); an oil phase containing Freund's type of adjuvant such as Freund's incomplete adjuvant (FIA); such as saponins Sapogenin glycosides; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine (commonly known as muramyl dipeptide or “MDP”); and lipopolysaccharide (LPS). Such adjuvants are generally used in the form of an emulsion with an aqueous phase or more generally may consist of a water-insoluble inorganic salt. These inorganic salts may comprise, for example, aluminum hydroxide, zinc sulfate, iron hydroxide colloid, calcium phosphate or calcium chloride. Aluminum hydroxide (Al (OH) ₃ ) is a commonly used adjuvant. Currently, the only FDA approved adjuvant for use in humans is the aluminum salt (alum), which is used to “reserve” antigen by precipitation of the antigen. The adjuvant provided above is merely illustrative. In practice, any adjuvant can be used in the SNAP-25 compositions disclosed herein as long as the adjuvant satisfies the essential characteristics for inducing an immune response.

  The carriers disclosed herein can also act as adjuvants. Specific adjuvants and methods of making and using are described, for example, in Gupta et al. Vaccine, 11: 993-306 (1993); Arnon, R. (Ed.) Synthetic Vaccines 1: 83-92, CRC Press, Inc., Boca Raton , Fla., (1987); and "Immunogenicity enhancing carriers and compositions thereof and methods of using the same" David W. Waggoner, Jr. et al., US Patent Application Publication No. 2004/0057958 (March 2004). 25th). Additional adjuvants include any of those described in Chapter 7 (pp 141-227) “Vaccine Design, The Subunit and Adjuvant Approach” (eds. Powell, MF and Newman, MJ) Pharmaceutical Biotechnology, Volume 6, Plenum Press (New York). These compounds are included. Examples from this introduction include muramyl dipeptide (MDP) and Montanide 720. A molecule such as polyinosine: cytosine (poly I: C) or plasmid DNA containing a CpG motif can also be administered in combination with an antigen encapsulated in microparticles as an adjuvant. In another example, the adjuvant is an agent that promotes entry of the antigenic compound into the cell cytoplasm, such as listeriolysin, streptricin, or mixtures thereof.

  Thus, in an embodiment, a SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated carboxyl terminal glutamine linked to a carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxyl terminal glutamine is SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37. SEQ ID NO: 147 or SEQ ID NO: 148. In another aspect of this embodiment, the SNAP-25 antigen comprises SEQ ID NO: 38. In aspects of this embodiment, the carrier peptide is keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP) ).

  In another embodiment, the SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated carboxyl terminal lysine linked to a carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxyl terminal lysine is SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. Comprising. In another aspect of this embodiment, the SNAP-25 antigen comprises SEQ ID NO: 45. In aspects of this embodiment, the carrier peptide is keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP) It is.

  In yet another embodiment, the SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to one or more mobile linkers and a carrier peptide, wherein the mobile The sex linker is interposed between the SNAP-25 antigen and the carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxyl terminal glutamine is SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37. SEQ ID NO: 147 or SEQ ID NO: 148. In another embodiment, the SNAP-25 antigen comprises SEQ ID NO: 46. In aspects of this embodiment, the carrier peptide is keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP) It is. In aspects of this embodiment, the mobile linker is a G spacer or an A spacer.

  In yet another embodiment, the SNAP-25 composition comprises a SNAP-25 antigen having a carboxylated C-terminal lysine linked to a mobile linker and a carrier peptide, wherein the mobile linker is a SNAP-25. Intervenes between the antigen and the carrier peptide. In aspects of this embodiment, the SNAP-25 antigen having a carboxylated carboxyl terminal lysine is SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. Comprising. In another aspect of this embodiment, the SNAP-25 antigen comprises SEQ ID NO: 47. In aspects of this embodiment, the carrier peptide is keyhole limpet hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI) or multiple attachment peptide (MAP) It is. In aspects of this embodiment, the mobile linker is a G spacer or an A spacer.

Aspects of the present disclosure generate α-SNAP-25 antibodies that bind in part to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. A method for comprising. By a variety of methods well known to alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond in the art from the SNAP-25 cleavage product Can be generated. Specific protocols for making and using antibodies as well as detecting and measuring antibody binding specificity, binding affinity and binding avidity are known in the art. For example ANTIBODIES: (.. Edward Harlow & David Lane, eds, Cold Spring Harbor Laboratory Press, 2 nd ed 1998a) A LABORATORY MANUAL; and USING ANTIBODIES: A LABORATORY MANUAL: PORABLE PROTOCOL No. I (Edward Harlow & David Lane, Cold Spring Harbor Laboratory Press, 1998b); Molecular Cloning, A Laboratory Manual, 2001; and Current Protocols in Molecular Biology, 2004; "Therapeutic polypeptides, nucleic acids encoding the same and methods of use" David Anderson et al. 7034132 (April 25, 2005); and “Antibodies to CTLA4” Beatriz M. Carreno et al., US Pat. No. 7034121 (April 25, 2006).

Non-limiting examples include SNAP-25 cleavage generation by injecting an animal, such as a rabbit, goat, mouse or another mammal, with one or more injections of the compositions disclosed herein. it is possible to generate a BoNT / a cleavage site scissile alpha-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the P ₁ residue from binding from the object. As another non-limiting example, BoNT / A cleavage from a SNAP-25 cleavage product by injecting an egg, such as a chicken egg, with one or more injections of the compositions disclosed herein. An α-SNAP-25 polyclonal antibody can be generated that binds to an epitope comprising the carboxyl terminus at the P ₁ residue from the site cleavable linkage. Antibody titers in immunized animals can be monitored over time by standard techniques such as using enzyme-linked immunosorbent assay (ELISA) or cell-based activity assay using immobilized antigen. A polyclonal antibody relating to an α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product, if desired, can be obtained in mammals (eg blood From) and can be further purified by well known techniques such as protein A affinity chromatography to obtain IgG fractions, or by affinity purification on the peptides used to generate the antibodies.

As another non-limiting example, α-SNAP-binding to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product using the hybridoma method. 25 monoclonal antibodies can be generated. See, eg Chapter 6 Monoclonal Antibodies, pp. 196-244, Harlow & Lane, supra, 1998a; and Chapter 7 Growing Hybridomas, pp. 245-282, Harlow & Lane, supra, 1998a; and Goding, pp. 59-103, Monoclonal. Antibodies: See Principles and Practice, Academic Press, (1986). In this way, for example, a mouse, a host animal such as a hamster, or another appropriate host animal, specifically bind to SNAP-25 having a carboxyl-terminus at the P ₁ residue of the BoNT / A cleavage site scissile bond Typically, one or more injections of the SNAP-25 antigen disclosed herein to elicit lymphocytes that will produce or are capable of producing alpha-SNAP-25 antibodies To experience. Antibody titers in immunized animals can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized antigen or cell-based activity assays. Alternatively, lymphocytes can be immunized in vitro using an appropriate cell culture system. Antibody producing cells are isolated from the animal at an appropriate time after immunization, for example when the antibody titer is highest. In general, peripheral blood lymphocytes are used when cells of human origin are desired, or spleen cells or lymph node cells are used when non-human mammalian sources are desired. Antibody-producing cells isolated using a suitable fusing agent such as polyethylene glycol are fused with an immortal cell line to form hybridoma cells. Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically, murine myeloma cell lines are fused with splenocytes taken from appropriately immunized mice to produce hybridomas. A preferred immortal cell line is a mouse myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT). Many myeloma cell lines such as P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines can be used as fusion partners by standard techniques. it can. Hybridoma cells that are the result of fusion using HAT medium which then kills unfused or non-productively fused myeloma cells (unfused splenocytes are not transformed and die after a few days) Select. Then the BoNT / A cleavage site scissile alpha-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at _{the P 1} residue from the binding of a culture medium in which hybridoma cells are growing from SNAP-25 cleavage product Can be tested for presence. Hybridoma supernatants using α-SNAP-25 positive media, for example in in vivo binding assays such as immunoprecipitation assays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA), or in cell-based activity assays, Can be screened. Such techniques and assays are known in the art. For example, Chapter 11 Immunoprecipitation, pp. 421-470, Harlow & Lane, supra, 1998a; Chapter 12 Immunoblotting, pp. 471-510, Harlow & Lane, supra, 1998a; Chapter 14 Immunoassays, pp. 553-612, Harlow & Lane , supra, 1998a. Then performing further studies, the antibody also may determine whether a non-reactive to SNAP-25 lacking a carboxyl-terminus at the P ₁ residue of the BoNT / A cleavage site scissile bond. The binding affinity of the α-SNAP-25 monoclonal antibody can also be determined, for example, by Scatchard analysis. See, e.g., "Multipurpose computer studies for the characterization of ligand: ligand binding systems" Peter J. Munson and David Rodbard, Anal. Biochem. 107 (1): 220-239 (1980). After identifying the desired hybridoma cells, a clone from a single cell is isolated until a clone cell line expressing the desired monoclonal antibody is obtained using a limiting dilution procedure. BoNT / A are cut sufficiently selective with respect to SNAP-25 having a carboxyl-terminus at the P ₁ residue of the site scissile bond, and select those antibodies that bind with sufficiently high avidity, further characterization and study .

Another alternative for preparing an α-SNAP-25 monoclonal antibody that binds to an epitope comprising the carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product is: for example a recombinant combinatorial immunoglobulin library, such as an antibody phage display library by screening with SNAP-25 peptide and the SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond Isolate immunoglobulin library members that bind. Kits for creating and screening phage display libraries are commercially available, eg, recombinant phage antibody systems (Amersham GE Healthcare, Piscataway, NJ); and SurfZAP ™ phage display kits (Stratagene, La Jolla, Calif.). It is available. In addition, illustrative examples of methods and reagents useful for generating and screening antibody display libraries include, for example, Ladner et al., US Pat. No. 5,223,409; Borrebaeck et al., US Pat. No. 5,712,089; Griffiths et al., US Pat. No. 5,885,793; Griffiths et al. McCafferty et al., US Pat. No. 5,969,108; Griffiths et al., US Pat. No. 6010884; Jespers et al., US Pat. No. 6,017,732; Borrebaeck et al., US Pat. No. 6,027,930; No .; McCafferty et al., US Pat. No. 6,172,197, each of which is hereby incorporated by reference in its entirety.

Aspects of the disclosure comprise, in part, collecting a sample containing α-SNAP-25 antibody or α-SNAP-25 antibody-producing cells. As used herein, the term “sample containing α-SNAP-25 antibody or α-SNAP-25 antibody-producing cell” refers to a BoNT / A cleavage site cleavable binding from a SNAP-25 cleavage product. Refers to any biological material that contains or potentially contains at least one α-SNAP-25 antibody that binds to an epitope comprising the carboxyl terminus at P ₁ residue. An α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product can include, but is not limited to: It is envisioned that any and all samples including blood, plasma, serum and lymph can be used in this method. Capable of generating an alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product, limiting However, it is also envisioned that any cell, including CD8 cells, CTL cells, helper T cells and B cells can be used in this method. Samples containing α-SNAP-25 antibody or α-SNAP-25 antibody producing cells can be collected from an individual using a variety of well-known methods, such as Harlow & Lane, supra, 1998a; and Harlow & Lane, See supra, 1998b. Similarly, α-SNAP− that binds to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product using a variety of well-known methods. Samples can be processed to isolate 25 antibodies. A procedure for collecting the sample can be selected based on the type of antibody to be isolated. Nonlimiting examples include isolating alpha-SNAP-25 polyclonal antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product In some cases, a suitable sample may be a blood sample containing such an α-SNAP-25 antibody, but containing a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. When isolating an α-SNAP-25 monoclonal antibody that binds to the epitope of interest, a suitable sample may be an α-SNAP-25 antibody producing cell such as a spleen cell or a hybridoma.

Aspects of the present disclosure include, in part, an α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at a P ₁ residue from a BoNT / A cleavage site cleavable bond from a SNAP-25 cleavage product from a sample. Isolating. For example SNAP-25 from the BoNT / A cleavage site scissile alpha-SNAP-25 polyclonal antibody in _{the P 1} residue from binding to bind an epitope comprising a carboxyl-terminus or SNAP-25 cleavage product from the cleavage product Methods of isolating such α-SNAP-25 antibodies, such as α-SNAP-25 monoclonal antibodies that bind to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage, are known to those skilled in the art. Is well known. See, for example, Harlow and Lane, supra, 1998a; and Harlow and Lane, supra, 1998b. For example, such α-SNAP-25 polyclonal antibody can be isolated from the sample by well-known techniques such as affinity chromatography using protein A or protein G, for example, which primarily provides an IgG fraction of immune serum. it can. Subsequently, or alternatively, a specific SNAP-25 antigen is immobilized on a column or magnetic bead and carboxyl terminal at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. An α-SNAP-25 polyclonal antibody that binds to an epitope comprising can be purified by immunoaffinity chromatography. SNAP-25 and alpha-SNAP-25 monoclonal antibody that binds an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from the culture medium or ascites fluid from the cleavage products, for example, protein A Isolation by conventional immunoglobulin purification procedures such as Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Thus, in an embodiment, the method of generating an α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable linkage from a SNAP-25 cleavage product comprises the steps: (A) administering to an animal a composition comprising a SNAP-25 antigen having a carboxylated C-terminal glutamine linked to a carrier peptide; (b) an α-SNAP-25 antibody or α-SNAP-25 Collecting a sample containing antibody producing cells from the animal; and (c) isolating the α-SNAP-25 antibody component from the sample. In aspects of this embodiment, SNAP-25 cleavage alpha-SNAP-25 antibody that binds to _{the P 1} residue epitope comprising a carboxyl-terminus at from the BoNT / A cleavage site scissile bond from the product in the polyclonal antibody is there. In another aspect of this embodiment, SNAP-25 α-SNAP- 25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from a monoclonal It is an antibody. In a further aspect of this embodiment, SNAP-25 BoNT / A cleavage site scissile alpha-SNAP-25 monoclonal which binds to _{the P 1} residue epitope comprising a carboxyl-terminus at from bond from cleavage products produced The antibody is an IgG subtype. In other aspects of this embodiment, the SNAP-25 composition further comprises an adjuvant, such as, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

In another embodiment, a method of generating an α-SNAP-25 antibody that binds to an epitope comprising a carboxyl terminus at the P ₁ residue from a BoNT / A cleavage site cleavable linkage from a SNAP-25 cleavage product comprises: Step (a) administering a composition comprising a SNAP-25 peptide having a carboxylated C-terminal glutamine linked to a mobile linker and a carrier peptide to an animal, wherein the mobile linker is a SNAP-25 peptide (B) collecting a sample containing α-SNAP-25 antibody or α-SNAP-25 antibody-producing cells from the animal; and (c) α-SNAP-25 antibody from the sample. Isolating. In aspects of this embodiment, SNAP-25 cleavage alpha-SNAP-25 antibody that binds to _{the P 1} residue epitope comprising a carboxyl-terminus at from the BoNT / A cleavage site scissile bond from the product in the polyclonal antibody is there. In another aspect of this embodiment, SNAP-25 α-SNAP- 25 antibodies that bind an epitope comprising a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from a monoclonal It is an antibody. In a further aspect of this embodiment, SNAP-25 BoNT / A cleavage site scissile alpha-SNAP-25 monoclonal which binds to _{the P 1} residue epitope comprising a carboxyl-terminus at from bond from cleavage products produced The antibody is an IgG subtype. In other aspects of this embodiment, the SNAP-25 composition further comprises an adjuvant, such as, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), or polyvinyl alcohol (PVA).

Aspects of the present disclosure comprise alpha-SNAP-25 antibodies isolated selectively binding to SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond in some . The term “isolated” as used herein refers to the separation of a molecule from its natural environment by using human intervention. As used herein, the term “antibody” refers to a molecule that binds specifically to an antigen made by the immune system made in response to a particular antigen, and includes naturally occurring antibodies and Includes both non-naturally occurring antibodies. As used herein, the term “α-SNAP-25” is synonymous with “anti-SNAP-25” and refers to an antibody that binds to a SNAP-25 antigen. For example, antibodies can be polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies, Ig receptor-like cell binding antibodies, as long as the fragment exhibits the desired biological activity. , Linear antibodies, diabodies or minibodies, and single chain derivatives of the same. An antibody can be a V _H and V _L domain, as well as a light chain constant domain (C _L ) and heavy chain constant domain, C _H1 , C _H2 and C _H3 , or such as a Fab fragment, F (ab ′) ₂ fragment, Fc fragment, Fd It may be a full-length immunoglobulin molecule comprising an immunologically active fragment of a full-length immunoglobulin molecule, such as a fragment, Fv fragment. The antibody can be derived from any vertebrate species (eg, human, goat, horse, donkey, mouse, rat, rabbit or chicken) and of any type (eg, IgG, IgE, IgM, IgD and IgA), class (Eg IgA, IgD, IgE, IgG and IgM) or subclasses (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). For general disclosure regarding the structure of naturally occurring antibodies, non-naturally occurring antibodies and antigenic compound-binding fragments thereof, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York pp. 269-315 (1994); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995), each of which is incorporated herein by reference in its entirety.

Naturally occurring antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons and are composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, but the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain and the heavy chain variable domain.

Complete antigen recognition and antigen binding sites are contained within the variable domain of the antibody, ie, the Fv fragment. This fragment contains a dimer of one heavy chain variable domain (V _H ) and one light chain variable domain (V _L ) in tight non-covalent association. Each domain comprises four framework regions (FR), which are mostly in β-sheet configuration and are connected by three hypervariable regions, which form a loop connecting β-sheet structures, and β In some cases, a part of the sheet structure is formed. Each hypervariable region comprises an amino acid sequence corresponding to a complementarity determining region (CDR). Collectively it is a three-dimensional configuration of six CDR regions that define the antigen binding sites on the surface of the V _H -V _L dimer that confer antigen-binding specificity. For example, “Conformation of immunoglobulin hypervariable region” Cyrus Chothia, et al., Nature 342 (6252): 877-883 (1989); Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), each of which is incorporated herein by reference in its entirety. The constant domain of an antibody does not participate directly in binding the antibody to the antigen, but exhibits various effector functions such as antibody involvement in antibody-dependent cytotoxicity.

  A target antigen generally has one or more binding sites, also called epitopes, that are recognized by the antigen binding site formed by the CDRs. As used herein, an “epitope” is synonymous with an “antigenic determinant” and is capable of specifically binding to an immunoglobulin or T cell receptor, or otherwise a molecule Refers to a site on a target antigen, such as a peptide, polysaccharide or lipid-containing molecule, that interacts with. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen can have more than one corresponding antibody.

  Polyclonal antibodies refer to heterogeneous populations of antibody molecules that contain at least two antibodies capable of binding a specific antigen. By definition, a polyclonal antibody comprises two different antibodies that bind to at least two different epitopes. As used herein, the term “monoclonal antibody” refers to a substantially homogenous population of antibody molecules containing only one antibody capable of binding to a particular antigen. The individual antibodies comprising the population, ie the population, are identical except for possible naturally occurring variants that may be present in trace amounts. By definition, a monoclonal antibody binds to a single epitope. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal antibodies, each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they can be synthesized without contaminating with other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. . For example, monoclonal antibodies for use in accordance with the present disclosure can be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or can be made by recombinant DNA methods (eg: US patents). No. 4,816,567; U.S. Pat. No. 5,807,715). For example, monoclonal antibodies can be ligated using the techniques described in Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991). It can also be isolated from a rally.

Thus, in an embodiment, the α-SNAP-25 antibody is a heavy chain variable domain (V _H ) and light chain that selectively binds to SNAP-25 having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage. It comprises a variable domain (V _L ). In aspects of this embodiment, the heavy chain variable domain (V _H ) is SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, or SEQ ID NO: 82. In another aspect of this embodiment, the light chain variable domain (V _L ) is SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, or SEQ ID NO: 92.

In another embodiment, the α-SNAP-25 antibody is a heavy chain variable domain (V _H ) CDR1 region that selectively binds to SNAP-25 having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage. , CDR2 region, CDR3 region or any combination thereof. In aspects of this embodiment, the heavy chain variable domain (V _H ) CDR1 region is SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In another aspect of this embodiment, the heavy chain variable domain (V _H ) CDR2 region is SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. In yet another aspect of this embodiment, the heavy chain variable domain (V _H ) CDR3 region is SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 124.

In another embodiment, the α-SNAP-25 antibody is a light chain variable domain (V _L ) CDR1 region that selectively binds to SNAP-25 having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage. , CDR2 region, CDR3 region, or any combination thereof. In aspects of this embodiment, the light chain variable domain (V _L ) CDR1 region is SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: : 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In another aspect of this embodiment, the light chain variable domain (V _L ) CDR2 region is SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112. In yet another aspect of this embodiment, the light chain variable domain (V _L ) CDR3 region is SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

In yet another embodiment, alpha-SNAP-25 antibody specifically binds an epitope comprising a SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond. In aspects of this embodiment, the epitope comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 148. It becomes. In aspects of this embodiment, the epitope comprises SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

As disclosed above, BoNT / A surrounding the cleavage site sequence _{P 5 -P 4 -P 3 -P 2} -P 1 -P 1 present in _{SNAP-25 '-P 2' -P} 3 '- P ₄ '-P ₅ ' is indicated, and P ₁ -P ₁ 'represents a cleavable bond. During cleavage by BoNT / A, is consequently generated cleavage products _{P 5 -P 4 -P 3 -P 2} -P fragment and _{P 1} containing ₁ sequence _{'-P 2' -P 3 '-P} 4 It comprises a fragment containing '-P ₅ '. As used herein, "SNAP-25 cleavage BoNT / A cleavage site scissile alpha-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at _{the P 1} residue from binding from the product "the term that selectively bind to any SNAP-25 cleavage product fragment comprising the _{P 5 -P 4 -P 3 -P 2} -P 1 _{sequence, P 1 '-P 2' -P} 3 Any SNAP-25 cleavage product fragment comprising the '-P ₄ ' -P ₅ 'sequence or any SNAP-25 having an intact P ₁ -P ₁ ' cleavable bond at the BoNT / A cleavage site It refers to α-SNAP-25 antibody that does not bind. As used herein, the term “α-SNAP-25 ₁₉₇ antibody” refers to an antibody that selectively binds to SNAP-25 having a carboxyl terminal P ₁ residue corresponding to glutamine 197 of SEQ ID NO: 5. Point to. As used herein, the term “α-SNAP-25 ₂₀₄ antibody” refers to an antibody that selectively binds to SNAP-25 having a carboxyl terminal P ₁ residue corresponding to lysine 204 of SEQ ID NO: 16. Point to.

As used herein, the term “selective” refers to having a unique effect or influence or reaction in a unique manner or with a unique one. As used herein, the term “selectively binds” when referring to an antibody refers to an antibody to the indicated target epitope such that the antibody does not substantially cross-react with the non-target epitope. Refers to the discriminative combination of. The minimum size peptide epitope as defined herein is about 5 amino acids and the peptide epitope is typically at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least It comprises 15 or at least 20 amino acids. A peptide epitope may be non-contiguous, i.e., it comprises amino acid residues that are not adjacent in the primary structure of the peptide but that together become an epitope due to the secondary, tertiary or quaternary structure of the peptide. In addition, the epitope may comprise a portion of a molecule other than an amino acid sequence, such as a carbohydrate moiety, a lipoprotein or glycolipid-like lipid moiety, or a phosphorylated amino acid-like chemically modified amino acid moiety. Also noted. In aspects of this embodiment, the α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, comprising at least 15, or at least 20 amino acids, optionally in the SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond Can be combined. In other aspects of this embodiment, BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of at most 5, A BoNT / A cleavage site cleavable bond comprising at most 6, at most 7, at most 8, at most 9, at most 10, at most 15 or at most 20 amino acids it can be a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of selectively bind.

Selective binding includes binding properties such as binding affinity, binding specificity and binding avidity. See David J. King, Applications and Engineering of Monoclonal Antibodies, pp. 240 (1998). Binding affinity refers to the length of time an antibody stays at its epitope binding site and can be considered as the strength with which the antibody binds to its epitope. The binding affinity can be described as the equilibrium dissociation constant (KD) of the antibody, which is defined as the Kd / Ka ratio at equilibrium. Where Ka is the association rate constant of the antibody and kd is the dissociation rate constant of the antibody. Binding affinity is determined by both association and dissociation, and high affinity or low dissociation alone cannot ensure high affinity. Association rate constant (Ka) or on-rate constant (Kon) measures the number of binding events per unit time, or the tendency of an antibody and antigen to reversibly associate with its antibody-antigen complex. . The association rate constant is expressed as M ⁻¹ s ⁻¹ and symbolically as follows: [Ab] × [Ag] × Kon. The higher the association rate constant, the more rapidly the antibody binds to its antigen or the higher the binding affinity between the antibody and antigen. The dissociation rate constant (Kd) or the off-rate constant (Koff) is the number of dissociation events per unit time, and the antibody-antigen complex is reversibly separated (dissociated) into its constituent molecules, ie, antibody and antigen. ) Measure the tendency to. The dissociation rate constant is expressed as s ⁻¹ and is expressed symbolically as follows: [Ab + Ag] × Koff. The smaller the dissociation rate constant, the more tightly the antibody binds to its antigen or the higher the binding affinity between the antibody and antigen. The equilibrium dissociation constant (KD) measures the rate at which the new antibody-antigen complex formed equals the rate at which the antibody-antigen complex equilibrates and dissociates. The equilibrium dissociation constant is expressed in M and Koff / Kon = [Ab] × [Ag] / [Ab + Ag] (where [Ab] is the molar concentration of the antibody and [Ag] is the molar concentration of the antigen. And [Ab + Ag] is the molar concentration of the antibody-antigen complex, where all concentrations are such components when the system is in equilibrium, the smaller the equilibrium dissociation constant, the more the antibody Binds more tightly or has a higher binding affinity between the antibody and the antigen.

Thus, in an embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is, for example, 1 × 10 ⁵ M ^{−. It} may have an association rate constant of less than ¹ s ^−1, less than 1 × 10 ⁶ M ⁻¹ s ^−1, less than 1 × 10 ⁷ M ⁻¹ s ⁻¹ , or less than 1 × 10 ⁸ M ⁻¹ s ^−1. . In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is, for example, 1 × 10 ⁵ M. Association rate constant greater than ⁻¹ s ⁻¹ , greater than 1 × 10 ⁶ M ⁻¹ s ⁻¹ , greater than 1 × 10 ⁷ M ⁻¹ s ⁻¹ , or greater than 1 × 10 ⁸ M ⁻¹ s ⁻¹ Can have. In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is 1 × 10 ⁵ M ^−1. s ⁻¹ to 1 × 10 ⁸ M ⁻¹ s ⁻¹ , 1 × 10 ⁶ M ⁻¹ s ⁻¹ to 1 × 10 ⁸ M ⁻¹ s ⁻¹ , 1 × 10 ⁵ M ⁻¹ s ⁻¹ to 1 × It may have an association rate constant between 10 ⁷ M ⁻¹ s ⁻¹ or between 1 × 10 ⁶ M ⁻¹ s ⁻¹ and 1 × 10 ⁷ M ⁻¹ s ⁻¹ .

In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is 1 × 10 ⁻³ s. ^It may have a dissociation rate constant of less than ^−1, less than 1 × 10 ⁻⁴ s ⁻¹ or less than 1 × 10 ⁻⁵ s ⁻¹ . In other aspects of this embodiment, BoNT / A cleavage site scissile bond _{the P 1} residue in binding affinity of alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus of example 1. Less than 0 × 10 ⁻⁴ s ^−1, less than 2.0 × 10 ⁻⁴ s ^−1, less than 3.0 × 10 ⁻⁴ s ^−1, less than 4.0 × 10 ⁻⁴ s ⁻¹ , 5.0 × Less than 10 ⁻⁴ s ^−1, less than 6.0 × 10 ⁻⁴ s ^−1, less than 7.0 × 10 ⁻⁴ s ^−1, less than 8.0 × 10 ⁻⁴ s ^−1, or 9.0 × 10 ^{− It} may have a dissociation rate constant of less than ⁴ s ⁻¹ . In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is, for example, 1 × 10 ^−3. It may have a dissociation rate constant greater than s ⁻¹ , greater than 1 × 10 ⁻⁴ s ⁻¹ , or greater than 1 × 10 ⁻⁵ s ⁻¹ . In other aspects of this embodiment, BoNT / A cleavage site scissile bond _{the P 1} residue in binding affinity of alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus of example 1. ^Greater than 0 × 10 ⁻⁴ s ⁻¹ , greater than 2.0 × 10 ⁻⁴ s ⁻¹ , greater than 3.0 × 10 ⁻⁴ s ⁻¹ , greater than 4.0 × 10 ⁻⁴ s ⁻¹ , Greater than 5.0 × 10 ⁻⁴ s ⁻¹ , greater than 6.0 × 10 ⁻⁴ s ⁻¹ , greater than 7.0 × 10 ⁻⁴ s ⁻¹ and 8.0 × 10 ⁻⁴ s ⁻¹ It may have a dissociation rate constant greater than or greater than 9.0 × 10 ⁻⁴ s ⁻¹ .

In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage has an equilibrium of less than 0.500 nM. It may have a dissociation constant. In aspects of this embodiment, BoNT / A cleavage site scissile bond SNAP-25 epitope binding affinity of alpha-SNAP-25 antibody that selectively binds with a carboxyl-terminus at _{the P 1} residue of such as less than 0.500nM Less than 0.450 nM, less than 0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200 nM, less than 0.150 nM, less than 0.100 nM or less than 0.050 nM. Can have. In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable bond is an equilibrium greater than 0.500 nM. It may have a dissociation constant. In aspects of this embodiment, BoNT / A cleavage site scissile bond SNAP-25 epitope binding affinity of alpha-SNAP-25 antibody that selectively binds with a carboxyl-terminus at _{the P 1} residue of the 0.500nM example More than 0.450 nM more than 0.400 nM more than 0.350 nM more than 0.300 nM more than 0.250 nM more than 0.200 nM more than 0.150 nM more than 0.100 nM Or it may have an equilibrium dissociation constant greater than 0.050 nM.

In yet another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is an intact SNAP- For example, less than 1 × 10 ⁰ M ⁻¹ s ^−1, less than 1 × 10 ¹ M ⁻¹ s ^−1, less than 1 × 10 ² M ⁻¹ s ^−1, less than 1 × 10 ³ M ⁻¹ s ⁻¹ Or an association rate constant of less than 1 × 10 ⁴ M ⁻¹ s ⁻¹ . In another embodiment, the binding affinity of an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is relative to intact SNAP-25. For example, at most 1 × 10 ⁰ M ⁻¹ s ⁻¹ , at most 1 × 10 ¹ M ⁻¹ s ⁻¹ , at most 1 × 10 ² M ⁻¹ s ⁻¹ , at most 1 × 10 ³ It may have an association rate constant of M ⁻¹ s ⁻¹ or at most 1 × 10 ⁴ M ⁻¹ s ⁻¹ .

Binding specificity is the ability of an antibody to distinguish between a molecule containing that epitope and a molecule that does not contain that epitope. One way to measure binding specificity is to compare the antibody Kon association rate for a molecule containing the epitope relative to the antibody Kon association rate for the molecule that does not contain the epitope. For example, the association rate constant (Ka) of an α-SNAP-25 antibody for a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable bond, eg, P Relative to a SNAP-25 that does not contain that epitope, such as a SNAP-25 epitope lacking the carboxyl terminus at _one residue or a SNAP-25 epitope with an intact -P ₁ 'cleavable bond at the BoNT / A cleavage site. To compare. In aspects of this embodiment, BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of, the epitope (s) Regarding SNAP-25 not included, for example, less than 1 × 10 ⁰ M ⁻¹ s ^−1, less than 1 × 10 ¹ M ⁻¹ s ^−1, less than 1 × 10 ² M ⁻¹ s ⁻¹ , 1 × 10 ³ M ⁻¹ It has an association rate constant (Ka) of less than s ⁻¹ , or less than 1 × 10 ⁴ M ⁻¹ s ⁻¹ . In other aspects of this embodiment, BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of its (s) For SNAP-25 without an epitope, for example, at most 1 × 10 ⁰ M ⁻¹ s ⁻¹ , at most 1 × 10 ¹ M ⁻¹ s ⁻¹ , at most 1 × 10 ² M ⁻¹ s ^−1. And an association rate constant (Ka) of at most 1 × 10 ³ M ⁻¹ s ⁻¹ or at most 1 × 10 ⁴ M ⁻¹ s ⁻¹ .

In a still further aspect of this embodiment, alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond can not contain the epitope Relative to SNAP-25, for example, at least 2 times more, at least 3 times more, at least 4 times more, at least 5 times more, at least 6 times more, at least 7 times more, at least 8 times more, or at least 9 with respect to that epitope Has twice as many association rate constants (Ka). In a further aspect of this embodiment, an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is a SNAP that does not contain that epitope. Relative to -25, for example, it has an association rate constant (Ka) that is at least 10-fold, at least 100-fold, at least 1,000-fold, or at least 10,000-fold greater for that epitope. In yet other aspects of this embodiment, alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond, contain the epitope Relative to no SNAP-25, for example, at most 1 times, at most 2 times, at most 3 times, at most 4 times, at most 5 times, at most Also have an association rate constant (Ka) of 6 times, at most 7 times, at most 8 times, or at most 9 times. In yet other aspects of this embodiment, alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond, contain the epitope Relative to no SNAP-25, for example, at most 10 times, at most 100 times, at most 1,000 times, or at most 10,000 times the association rate constant (Ka) for that epitope. ).

BoNT / in _{the P 1} residue of the A cleavage site scissile bond to SNAP-25 epitope having a carboxyl-terminus of the binding specificity of alpha-SNAP-25 antibody that selectively binds, for example BoNT / A cleavage site scissile bond P ₁ residues such as SNAP-25 epitope having an intact _P 1 -P ₁ 'scissile bond SNAP-25 epitope lacking a carboxyl-terminus or BoNT / a cleavage site, SNAP-25, which does not contain the epitope In contrast, such an α-SNAP-25 antibody can also be characterized as a ratio that can distinguish its SNAP-25 epitope. In an aspect of this embodiment, an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable linkage is a SNAP- that does not contain that epitope. For example, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 64: 1, at least 7: 1, at least 8: 1, at least 9 with respect to its SNAP-25 epitope. Having a binding specificity ratio of: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1 or at least 40: 1. In yet other aspects of this embodiment, BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of, BoNT / A cleavage Relative to SNAP-25, which lacks the carboxyl terminus at the P ₁ residue of the site cleavable linkage, for example for its SNAP-25 epitope at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, At least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1 or Have a binding specificity ratio of at least 40: 1. In still other aspects of this embodiment, BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of, BoNT / A cleavage Relative to a SNAP-25 having an intact P ₁ -P ₁ ′ cleavable bond at the site, for example, at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1 with respect to its SNAP-25 epitope At least 64: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1 or Have a binding specificity ratio of at least 40: 1.

Binding avidity, also known as functional affinity, refers to the sum of functional binding forces between a multivalent antibody and its antigen. Antibody molecules can have more than one binding site (eg, 2 for IgG, 10 for IgM), and many antigens contain more than 1 antigenic site. The binding avidity of an antibody depends on the binding affinity of the individual antibody binding sites, but since all antibody-antigen interactions must be destroyed at the same time for the antibody to completely dissociate, the binding avidity is greater than the binding affinity. Is also big. BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at _{the P 1} residue of selectively bind to any and all epitopes for that antibody It is envisaged that it can be done.

Thus in embodiments, alpha-SNAP-25 antibody is alpha-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond. In aspects of this embodiment, the α-SNAP-25 antibody is selective for an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl-terminal glutamine, or a SNAP-25 epitope having a carboxyl-terminal lysine. Α-SNAP-25 antibody that binds to In another aspect of this embodiment, the α-SNAP-25 antibody selectively binds to an SNAP-25 epitope having a carboxyl terminal P ₁ residue corresponding to glutamine 197 of SEQ ID NO: 5. Or an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminal P ₁ residue corresponding to lysine 204 of SEQ ID NO: 16. In yet another aspect of this embodiment, the α-SNAP-25 antibody comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: : 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, SEQ ID NO: 147 or SNAP-25 epitopes having the carboxyl terminal amino acid sequence of SEQ ID NO: 148 Is an α-SNAP-25 antibody that binds naturally.

  Aspects of the present disclosure comprise in part an immune-based method for detecting BoNT / A activity. The immune-based methods disclosed herein can be used, for example, for accuracy, accuracy, limit of detection (LOD), limit of quantification (LOQ), range, specificity, selectivity, linearity, durability and system suitability. It can be evaluated by several parameters including: The accuracy of the method is a measure of the accuracy of the analytical method, or the closeness of agreement between the measured value and the value accepted as a conventional true value or an accepted reference value. The accuracy of the method is the degree of agreement between the individual test results when the procedure is repeatedly applied to multiple samplings of a uniform sample. As such, accuracy is 1) intra-assay variability; 2) intra-day variability (concurrent accuracy); and 3) inter-day variability (indoor reproducibility); and 4) interlaboratory variability (inter-room reproducibility); To evaluate. The coefficient of variation (CV%) is a quantitative measure of accuracy expressed relative to the observed or theoretical average.

The immuno-based method disclosed herein, comprising a SNAP-25 having a carboxyl-terminus above background in _{the P 1} residue of the BoNT / A cleavage site scissile bond alpha-SNAP-25 antibody - It must be possible to detect the presence of the antigen complex. The limit of detection (LOD) of the method refers to the concentration of analyte that raises a signal that is significantly different from the negative control or blank, and represents the lowest concentration of analyte that can be distinguished from the background.

  Thus, in an embodiment, the immuno-based methods disclosed herein can detect BoNT / A LOD in an amount significantly different from a negative control or blank. In aspects of this embodiment, the immune-based methods disclosed herein are, for example, 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, 1 ng or less. Has a BoNT / A LOD. In yet another aspect of this embodiment, the immune-based methods disclosed herein are, for example, 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, 100 pg or less. Has a BoNT / A LOD. In a further aspect of this embodiment, the immune-based methods disclosed herein are, for example, 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, 10 pg or less BoNT / A LOD. In other aspects of this embodiment, the immune-based methods disclosed herein may include, for example, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less BoNT. / A LOD. In still other aspects of this embodiment, the immune-based methods disclosed herein are, for example, 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.5 pg or less. It has a BoNT / A LOD of 4 pg or less, 0.3 pg or less, 0.2 pg or less, 0.1 pg or less.

  In another aspect of this embodiment, the immune-based methods disclosed herein are, for example, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM. It has the following BoNT / A LOD. In other aspects of this embodiment, the immune-based methods disclosed herein are, for example, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less BoNT. / A LOD. In other aspects of this embodiment, the immuno-based methods disclosed herein can be, for example, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM. It has the following BoNT / A LOD. In still other aspects of this embodiment, the immuno-based methods disclosed herein comprise 10 pM or less BoNT / A, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less, It has a BoNT / A LOD of 2 pM or less or 1 pM or less. In yet another aspect of this embodiment, the immune-based methods disclosed herein can be, for example, 1,000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM. Or a BoNT / A LOD of 100 fM or less. In yet another aspect of this embodiment, the immune-based methods disclosed herein can be, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or It has a BoNT / A LOD of 10 fM or less. In yet another aspect of this embodiment, the immuno-based methods disclosed herein can be, for example, 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or Botulinum neurotoxin A LOD of 1 fM or less.

  The limit of quantification (LOQ) is the lowest and highest concentration of analyte in a sample or specimen that can be measured with an acceptable level of accuracy and precision. The lower limit of quantification refers to the lowest dose that the detection method can consistently measure from the background. The upper limit of quantification is the highest dose that the detection method can consistently measure from the background before signal saturation occurs. The range of method linearity is the area between the lower and upper quantitation limits. The linearity range is calculated by subtracting the lower quantitation limit from the upper quantitation limit. As used herein, the term “signal to noise ratio for the lower asymptote” refers to the signal detected in the method at the lower detection limit divided by the background signal. As used herein, the term “signal to noise ratio for the upper asymptote” refers to the signal detected in the method at the upper detection limit divided by the background signal.

  Thus, in an embodiment, the immuno-based methods disclosed herein are capable of detecting BoNT / A LOQ in amounts significantly different from negative controls or blanks. In aspects of this embodiment, the immune-based methods disclosed herein are, for example, 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, 1 ng or less. It has a BoNT / A LOQ. In yet another aspect of this embodiment, the immune-based methods disclosed herein are, for example, 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, 100 pg or less. It has a BoNT / A LOQ. In a further aspect of this embodiment, the immune-based methods disclosed herein are, for example, 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, 10 pg or less BoNT / A has a LOQ. In other aspects of this embodiment, the immune-based methods disclosed herein may include, for example, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less BoNT. / A LOQ. In still other aspects of this embodiment, the immune-based methods disclosed herein are, for example, 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.5 pg or less. It has a BoNT / A LOQ of 4 pg or less, 0.3 pg or less, 0.2 pg or less, 0.1 pg or less.

  In another aspect of this embodiment, the immune-based methods disclosed herein are, for example, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM. It has the following BoNT / A LOQ. In other aspects of this embodiment, the immune-based methods disclosed herein are, for example, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less BoNT. / A LOQ. In other aspects of this embodiment, the immuno-based methods disclosed herein can be, for example, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM. It has the following BoNT / A LOQ. In still other aspects of this embodiment, the immuno-based methods disclosed herein may include, for example, 10 pM or less BoNT / A, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less. It has a BoNT / A LOQ of 2 pM or less or 1 pM or less. In yet another aspect of this embodiment, the immune-based methods disclosed herein can be, for example, 1,000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM. Or a BoNT / A LOQ of 100 fM or less. In yet another aspect of this embodiment, the immune-based methods disclosed herein can be, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or It has a BoNT / A LOQ of 10 fM or less. In yet another aspect of this embodiment, the immuno-based methods disclosed herein can be, for example, 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or It has a BoNT / A LOQ of 1 fM or less.

  An immuno-based assay useful for practicing the disclosed method embodiment should have an accuracy of only 50%. In aspects of this embodiment, the immune-based assay has an accuracy of only 50%, only 40%, only 30% or only 20%. In other aspects of this embodiment, the immune-based assay has an accuracy of only 15%, only 10%, or only 5%. In other aspects of this embodiment, the immune-based assay has an accuracy of only 4%, only 3%, only 2% or only 1%.

  An immune-based assay useful for practicing the disclosed method aspects should have an accuracy of at least 50%. In aspects of this embodiment, the immune-based assay has an accuracy of at least 50%, at least 60%, at least 70% or at least 80%. In other aspects of this embodiment, the immune-based assay has an accuracy of at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the immune-based assay has an accuracy of at least 96%, at least 97%, at least 98%, or at least 99%.

  The immune-based methods disclosed herein must have a signal-to-noise ratio for the lower asymptote that is statistically significant and a signal-to-noise ratio for the upper asymptote that is statistically significant. In aspects of this embodiment, the immune-based methods disclosed herein are, for example, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1 with respect to the asymptote below. It has a signal to noise ratio of at least 8: 1, at least 9: 1, at least 10: 1, at least 15: 1 or at least 20: 1. In other aspects of this embodiment, the immune-based method is, for example, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 30: 1, at least 35: 1 with respect to the asymptote above. At least 40: 1, at least 45: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1 or at least 100: 1, at least 150: 1, at least 200: 1, It has a signal to noise ratio of at least 250: 1, at least 300: 1, at least 350: 1, at least 400: 1, at least 450: 1, at least 500: 1, at least 550: 1 or at least 600: 1.

  The specificity of the method defines the ability of the method to measure analytes that are important for the exclusion of other related components, such as, for example, partially active or inactive analytes. The selectivity of the method describes the ability of the analytical method to differentiate different substances in the sample. The linearity of the method is its ability to elicit results by a well-defined mathematical transformation that is either directly proportional to the concentration of the analyte in the sample. Thus, in an embodiment, the immuno-based methods disclosed herein may provide a fully active BoNT / A, such as 70% or less, 60% or less, 50% or less, It can be distinguished from partially active BoNT / A having no more than%, no more than 30%, no more than 20% or no more than 10%.

  The durability of the method is the room-to-room reproducibility of the test results obtained for the same sample under normal (but fluctuating) test conditions. The robustness of the procedure is a measure of its acceptability that is small in method parameters but unaffected by careful variation and provides an indication of its reliability in normal use. Therefore, durability evaluates changes that are difficult to avoid, but robustness evaluates careful changes. Typical parameters evaluated by durability and robustness include freeze / thaw, incubation time, incubation temperature, reagent lifetime, sample preparation, sample storage, cell passage number, many toxins, variability between purifications And the effect of variability between nicking reactions. Robustness parameters for cell-based assays include cell bank (beginning of freeze, middle and end), cell passage level, cell seeding density, cell stock density (days of culture), cell age in flask (until seeding) Waiting time), incubation time, various plates, overdose serum, and source of reagents. System suitability of the method is a determination of assay performance, including reagent and instrument performance, over time by analysis of reference standards. FDA guidance emphasizes system suitability with reference to the fact that equipment, electronics, assay performance and the sample to be analyzed constitute an integrated system. When plotting the log of dose versus response, system suitability can be assessed by testing for parallelism, where serial dilutions of the reference material and serial dilutions of the sample should produce parallel curves.

  Aspects of the disclosure comprise in part cells from established cell lines. As used herein, the term “cell” refers to any eukaryotic cell that is sensitive to or capable of taking up BoNT / A by BoNT / A poisoning. Point to. The term cell includes cells from various organisms such as, for example, murine, rat, pig, bovine, horse, primate and human cells; from various cell types such as neuronal and non-neuronal; And can be isolated from or part of a homogeneous cell population, tissue or organism. As used herein, the term “established cell line” is synonymous with “immortal cell line” or “transformed cell line” and is derived from an organism, tissue or organ source. Refers to a cell culture of cells selected for unlimited propagation from a cell population. By definition, established cell lines exclude primary cell cultures. As used herein, the term “primary cell” is a cell taken directly from fresh tissue or organ and does not have the potential to grow indefinitely. An established cell line can comprise a heterogeneous population of cells or a homogeneous population of cells. An established cell line derived from a single cell is referred to as a clonal cell line. An established cell line is one in which the cell is responsible for the overall cellular mechanism (by which BoNT / A cleaves the SNAP-25 substrate by proteolysis and binding of BoNT / A to the BoNT / A receptor, neurotoxin / Internalization of the receptor complex, translocation of BoNT / A light chain from intracellular vesicles to cytoplasm and proteolytic cleavage of SNAP-25) It may be expressed endogenously. Instead, an established cell line is one in which the cell does From exogenous sources to internalize the neurotoxin / receptor complex, translocate the BoNT / A light chain from the intracellular vesicles to the cytoplasm and proteolytic cleavage of SNAP-25) At least one component may be introduced. Cells from such established cell lines, also referred to as genetically engineered cell lines, can express, for example, exogenous FGFR2, exogenous FGFR3, exogenous SV2, exogenous SNAP-25, or any combination thereof.

  Aspects of the present disclosure comprise in part cells from established cell lines that are sensitive to BoNT / A poisoning. As used herein, “cell (s) sensitive to BoNT / A poisoning”, “cell (s) sensitive to BoNT / A poisoning by BoNT / A” or “BoNT / A” The term "cell (s) from an established cell line that is sensitive to BoNT / A intoxication by" refers to the overall cellular mechanism whereby BoNT / A cleaves the SNAP-25 substrate by proteolysis and BoNT Includes binding of A / A to BoNT / A receptor, internalization of neurotoxin / receptor complex, translocation of BoNT / A light chain from intracellular vesicle to cytoplasm and proteolytic cleavage of SNAP-25 Refers to the cell (s) that can be performed. By definition, cells (s) that are sensitive to BoNT / A poisoning express or must be engineered to express at least one BoNT / A receptor and at least one SNAP-25 substrate. . As used herein, “cell (s) capable of taking up BoNT / A” or “cell (s) comprising an established cell line capable of taking up BoNT / A”. The term is an overall cellular mechanism whereby BoNT / A cleaves the SNAP-25 substrate by proteolysis and binding of BoNT / A to the BoNT / A receptor, internalization of the neurotoxin / receptor complex , Including translocation of BoNT / A light chain from intracellular vesicles to cytoplasm and proteolytic cleavage of SNAP-25). By definition, the cell (s) that can take up BoNT / A must express or be engineered to express at least one BoNT / A receptor and at least one SNAP-25 substrate. .

  Thus, in an embodiment, cells from established cell lines are sensitive to BoNT / A poisoning. In aspects of this embodiment, cells from established cell lines are sensitive to BoNT / A poisoning by BoNT / A, for example, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less. In other aspects of this embodiment, cells from established cell lines are, for example, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less. Alternatively, it is highly sensitive to BoNT / A poisoning by BoNT / A of about 10 pM or less. In still other embodiments, cells from established cell lines are, for example, about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less or about 1 pM. It is sensitive to BoNT / A poisoning caused by the following BoNT / A. In still other embodiments, cells from established cell lines are, for example, about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less, about 0.4 pM. Hereinafter, it is highly sensitive to BoNT / A poisoning by BoNT / A of about 0.3 pM or less, about 0.2 pM or about 0.1 pM or less. As used herein, the term “about” is used to describe the value of a described item, percentage, parameter or term when the value of the described item, number, percentage or term is appropriate. + Or -10% range.

  In another embodiment, a cell comprising an established cell line can take up BoNT / A. In aspects of this embodiment, the cells comprising the established cell line can take up, for example, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less of BoNT / A. In other aspects of this embodiment, the cell comprising the established cell line is about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM. Possesses the ability to capture less or less than about 10 pM BoNT / A. In still other embodiments, the cell comprising the established cell line is about 9 pM or less, about 8 pM or less, about 7 pM or less, about 6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM or less, about 2 pM or less or about Has the ability to capture BoNT / A of 1 pM or less. In yet other embodiments, the cells comprising the established cell line are about 0.9 pM or less, about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about 0.5 pM or less, about 0.5 pM. Possesses the ability to capture BoNT / A of 4 pM or less, about 0.3 pM or less, about 0.2 pM or less, or about 0.1 pM or less.

  Aspects of the present disclosure in part comprise BoNT / A. As used herein, the term “BoNT / A” is synonymous with “botulinum neurotoxin serum type A” or “botulinum neurotoxin type A”, and naturally occurring BoNT / A or its non-naturally occurring It refers to both BoNT / A and includes a BoNT / A complex comprising about 150 kDa BoNT / A neurotoxin and related non-toxin related protein (NAP), and about 150 kDa BoNT / A neurotoxin alone. Non-limiting examples of BoNT / A complexes include, for example, the 900 kDa BoNT / A complex, the 500 kDa BoNT / A complex, and the 300 kDa BoNT / A complex. Non-limiting examples of BoNT / A neurotoxins of about 150 kDa include, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

  As used herein, the term “naturally-occurring BoNT / A” includes, but is not limited to, BoNT / A generated from post-translational modifications, alternatively spliced transcripts, or spontaneous mutations. Produced by naturally occurring processes including isoforms and BoNT / A subtypes such as BoNT / A1 subtype, BoNT / A2 subtype, BoNT / A3 subtype, BoNT / A4 subtype and BoNT / A5 subtype Refers to any BoNT / A. Naturally occurring BoNT / A includes, but is not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. Or from SEQ ID NO: 4, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more , 30 or more, 40 or more, 50 or more, or 100 amino acids substituted, deleted or added. Commercially available pharmaceutical compositions of naturally occurring BoNT / A include, but are not limited to, BOTOX® (Allergan, Inc., Irvine, Calif.), DYSPORT® / RELOXIN® ), (Ipsen Ltd., Slough, England), PURTOX (registered trademark) (Mentor Corp., Santa Barbara, CA), XEOMIN (registered trademark) (Merz Pharmaceuticals, GmbH., Frankfurt, Germany), NEURONOX (registered trademark) (Medy-Tox, Inc., Ochang-myeon, South Korea), BTX-A.

  As used herein, the term “non-naturally occurring BoNT / A” includes, but is not limited to, a modified amino acid generated by genetic engineering using random mutagenesis or rational design. It refers to any BoNT / A whose structure has been modified with the aid of human manual manipulation, including BoNT / A with sequences and BoNT / A produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring BoNT / A are, for example, “post-translational modifications and clostridial neurotoxins” Steward, LE et al., US Pat. No. 7,223,577; “activatable clostridial toxins” Dolly, JO et al., US Pat. No. 7,419,676. "Clostridial neurotoxin compositions and modified Clostridial neurotoxins" Steward, LE et al., US Patent Application Publication No. 2004/0220386; "Modified Clostridial toxins with enhanced targeting capabilities with respect to the endogenous Clostridial toxin receptor system" Steward, LE Et al., US Patent Application Publication No. 2008/0096248; "Modified Clostridial Toxins with Altered Targeting Capabilities for Clostridial Toxin Target Cells" Steward, LE et al., US Patent Application Publication No. 2008/0161543; Modified clostridial toxin with enhanced translocation ability and altered targeting activity with respect to target cells, "Steward, LE et al., US Patent Application Publication No. 2008/0241881, each of which is incorporated herein by reference in its entirety. Part).

  Thus, in an embodiment, the detected BoNT / A activity is derived from naturally occurring BoNT / A. In aspects of this embodiment, the detected BoNT / A activity is derived from a BoNT / A isoform or a BoNT / A subtype. In aspects of this embodiment, the detected BoNT / A activity is derived from BoNT / A of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In another aspect of this embodiment, the detected BoNT / A activity is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, for example at least 70%, at least 75%, at least 80% Derived from BoNT / A having at least 85%, at least 90% or at least 95% amino acid identity. In other aspects of this embodiment, the detected BoNT / A activity is BOTOX®, DYSPORT® / RELOXIN®, PURTOX®, XEOMIN®, NEURONOX ( (Registered trademark) or BTX-A.

  In another embodiment, the detected BoNT / A activity is derived from non-naturally occurring BoNT / A. In another aspect of this embodiment, the detected BoNT / A activity is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, for example at least 70%, at least 75%, at least 80% Derived from non-naturally occurring BoNT / A variants having at least 85%, at least 90% or at least 95% amino acid identity. In other aspects of this embodiment, the detected BoNT / A activity is, for example, one or more, two or more relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 Derived from a non-naturally occurring BoNT / A variant with one or more non-contiguous amino acid substitutions, deletions or additions. In yet another aspect of this embodiment, the detected BoNT / A activity is, for example, one or more, two or more relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or Derived from a non-naturally occurring BoNT / A variant with 100 or more contiguous amino acid substitutions, deletions or additions.

  Aspects of the present disclosure comprise SNAP-25 in part. As used herein, the term “SNAP-25” refers to naturally occurring SNAP-25 or non-naturally occurring SNAP-25 that is preferentially cleaved by BoNT / A. As used herein, the term “preferentially cleaved” means that the cleavage rate of BoNT / A substrate by BoNT / A is at least greater than the cleavage rate of any other substrate by BoNT / A. One digit higher. In aspects of this embodiment, the rate of cleavage of the BoNT / A substrate by BoNT / A is at least 2 orders of magnitude higher, at least 3 orders of magnitude higher, at least 4 orders of magnitude higher than the rate of cleavage of any other substrate by BoNT / A, or at least 5 digits higher.

  As used herein, the term “naturally-occurring SNAP-25” includes, but is not limited to, SNAP-25 generated from post-translational modifications, alternatively spliced transcripts or spontaneous mutations. Refers to any SNAP-25 produced by naturally occurring processes, including isoforms and SNAP-25 subtypes. Although not limited to naturally occurring SNAP-25, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 21 SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, or SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 are also included Or from SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 As described above, those in which 30 or more, 40 or more, 50 or more, or 100 or more amino acids are substituted, deleted or added are included.

  As used herein, the term “non-naturally occurring SNAP-25” includes, but is not limited to, SNAP-25 produced by genetic engineering using random mutagenesis or rational design, And any SNAP-25 whose structure has been modified with the aid of human manual manipulation, including SNAP-25 produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring SNAP-25 are, for example, “FRET Protease Assay for Clostridial Toxin” Steward, LE et al., US Pat. No. 7,332,567; “Lipophilic Dye-Based FRET Assay for Clostridial Toxin Activity” Fernandez-Salas et al. Patent Application Publication No. 2008/0160561, each of which is incorporated herein by reference in its entirety. Non-naturally occurring SNAP-25 is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 As described above, 30 or more, 40 or more, 50 or more, or 100 or more amino acids can be substituted, deleted or added.

  Thus, in an embodiment, SNAP-25 is a naturally occurring SNAP-25. In aspects of this embodiment, the SNAP-25 is a SNAP-25 isoform or a SNAP-25 subtype. In aspects of this embodiment, naturally occurring SNAP-25 is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 11. : 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: : 22, SEQ ID NO: 23 or SEQ ID NO: 24 naturally occurring SNAP-25. In another aspect of this embodiment, SNAP-25 comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 21 No. 22, SEQ ID NO: 23 or SEQ ID NO: 24, eg, naturally occurring with at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid identity SNAP-25.

  In another embodiment, the SNAP-25 is a non-naturally occurring SNAP-25. In other aspects of this embodiment, the SNAP-25 is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 with, for example, at least 70%, at least 75%, at least 80%, at least 85%, It is a non-naturally occurring SNAP-25 with at least 90% or at least 95% amino acid identity. In another aspect of this embodiment, SNAP-25 is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: : 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: : 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 A non-naturally occurring SNAP-25 having 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-adjacent amino acid substitutions, deletions or additions. In yet another aspect of this embodiment, SNAP-25 is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 11 SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 21 Number: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, A non-naturally occurring SNAP-25 having 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more adjacent amino acid substitutions, deletions or additions.

  The SNAP-25 may be endogenous SNAP-25 or exogenous SNAP-25. As used herein, the term “endogenous SNAP-25” refers to the genetic source encoding an external source of SNAP-25 or SNAP-25 since it is naturally encoded in the genome of the cell. Refers to SNAP-25 that is naturally present in a cell such that the cell naturally expresses SNAP-25 without the need for an external source of material. Endogenous SNAP-25 expression may or may not be accompanied by environmental stimuli such as cell differentiation. By definition, endogenous SNAP-25 may be only naturally occurring SNAP-25 or a variant thereof. For example, the following established cell lines express endogenous SNAP-25: BE (2) -M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH -SY5Y, SiMa and SK-N-BE (2) -C.

  As used herein, the term “exogenous SNAP-25” refers to the introduction of an external source of SNAP-25 or genetic material encoding SNAP-25 by human manual manipulation. Refers to SNAP-25 expressed in cells. The expression of exogenous SNAP-25 may or may not be accompanied by environmental stimuli such as cell differentiation. As a non-limiting example, cells from an established cell line can express exogenous SNAP-25 by transient or stable transfection of SNAP-25. As another non-limiting example, cells from an established cell line can express exogenous SNAP-25 by protein transfection of SNAP-25. Exogenous SNAP-25 may be naturally occurring SNAP-25 or a variant thereof, or non-naturally occurring SNAP-25 or a variant thereof.

  Thus, in an embodiment, cells from an established cell line express endogenous SNAP-25. In aspects of this embodiment, the endogenous SNAP-25 expressed by cells from the established cell line is a naturally occurring SNAP-25. In another aspect of this embodiment, endogenous SNAP-25 expressed by cells from an established cell line is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In yet a further aspect of this embodiment, the endogenous SNAP-25 expressed by cells from the established cell line is a naturally occurring SNAP-25 such as, for example, a SNAP-25 isoform or a SNAP-25 subtype. In another aspect of this embodiment, endogenous SNAP-25 expressed by cells from an established cell line is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least It is a naturally occurring SNAP-25 with 90% or at least 95% amino acid identity.

  In another embodiment, cells from established cell lines are engineered to transiently or stably express exogenous SNAP-25. In aspects of this embodiment, cells from established cell lines are engineered to transiently or stably express naturally occurring SNAP-25. In other aspects of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, engineered to express naturally occurring SNAP-25 of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In yet another aspect of this embodiment, cells from an established cell line transiently or stably express naturally occurring SNAP-25, such as a SNAP-25 isoform or a SNAP-25 subtype. To be operated. In yet another aspect of this embodiment, cells from the established cell line are transiently or stably expressed as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, sequence SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 18. SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, Engineered to express naturally occurring SNAP-25 having at least 90% or at least 95% amino acid identity.

  In another aspect of the embodiment, cells from established cell lines are engineered to transiently or stably express non-naturally occurring SNAP-25. In other aspects of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 and for example at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95 Engineered to express non-naturally occurring SNAP-25 with% amino acid identity. In other aspects of this embodiment, cells from the established cell line are transiently or stably expressed as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 8. : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: : 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 1 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-adjacent amino acid substitutions, missing To express non-naturally occurring SNAP-25 with loss or addition It is operated. In yet another aspect of this embodiment, cells from the established cell line are transiently or stably expressed as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, sequence SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 18. Number: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more adjacent amino acid substitutions, missing To express non-naturally occurring SNAP-25 with loss or addition It is operated.

  An assay that detects cleavage of the BoNT / A substrate following exposure to BoNT / A can be used to assess whether cells are expressing endogenous or exogenous SNAP-25. These assays detect the production of SNAP-25 cleavage products after BoNT / A treatment in cells expressing SNAP-25. Non-limiting examples of specific western blot analysis and well-characterized reagents, conditions and protocols include, but are not limited to, Amersham Biosciences, Piscataway, NJ; Bio-Rad Laboratories, Hercules, CA; Pierce Biotechnology, Inc., Rockford, IL; readily available from commercial manufacturers including Promega Corporation, Madison, WI; and Stratagene, Inc., La Jolla, CA. These and similar assays for SNAP-25 cleavage may be useful for identifying cells that express endogenous or exogenous SNAP-25.

  Non-limiting examples include assaying for BoNT / A incorporation using Western blot analysis using BoNT / A SNAP-25 cleavage products or antibodies that recognize both cleaved and uncleaved forms of SNAP-25. it can. Examples of α-SNAP-25 antibodies useful in these assays include, but are not limited to, α-SNAP-25 mouse monoclonal antibody SMI-81 (Sternberger Monoclonals Inc., Lutherville, MD), mouse α-SNAP. -25 monoclonal antibody CI71.1 (Synaptic Systems, Goettingen, Germany), α-SNAP-25 mouse monoclonal antibody CI71.2 (Synaptic Systems, Goettingen, Germany), α-SNAP-25 mouse monoclonal antibody SP12 (Abcam, Cambridge, Cambridge) MA), α-SNAP-25 rabbit polyclonal antiserum (Synaptic Systems, Goettingen, Germany), α-SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge, MA) and α-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma , St Louis, MO).

  Aspects of the disclosure comprise in part the BoNT / A receptor. As used herein, the term “BoNT / A receptor” refers to a naturally occurring BoNT / A receptor or non-naturally occurring that interacts preferentially with BoNT / A in a manner that elicits a BoNT / A toxic response. Refers to any of the developing BoNT / A receptors. As used herein, the term “preferentially interacts” refers to the BoNT / A equilibrium dissociation constant (KD) for the BoNT / A receptor, which relates to any other receptor on the cell surface. It is at least an order of magnitude smaller than that of BoNT / A. Equilibrium, which is a specific type of equilibrium constant that measures the tendency of the BoNT / A-BoNT / A receptor complex to reversibly separate (dissociate) into its component molecules, namely BoNT / A and BoNT / A receptors. The dissociation constant is defined as KD at equilibrium = Ka / Kd. The association constant (Ka) is defined as Ka = [C] / [L] [R] and the dissociation constant (Kd) is defined as Kd = [L] [R] / [C], where [L] Is equal to the molar concentration of BoNT / A, [R] is the molar concentration of BoNT / A receptor, and [C] is the molar concentration of BoNT / A-BoNT / A receptor complex, where All concentrations are of such components when the system is in equilibrium. The smaller the dissociation constant, the more tightly bound BoNT / A is to its receptor, or the higher the binding affinity between BoNT / A and BoNT / A receptor. In aspects of this embodiment, the BoNT / A dissociation constant for the BoNT / A receptor is at least 2 orders of magnitude, at least 3 orders of magnitude, at least 4 orders of magnitude less than that of BoNT / A for any other receptor, Or at least 5 orders of magnitude smaller. In another aspect of this embodiment, the binding affinity of BoNT / A that interacts preferentially with the BoNT / A receptor has an equilibrium dissociation constant (KD) of, for example, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less. Can have. In other aspects of this embodiment, the binding affinity of BoNT / A that interacts preferentially with the BoNT / A receptor is, for example, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM. Or an equilibrium dissociation constant (KD) of 10 nM or less. As used herein, the term “elicits a BoNT / A toxic response” means that the BoNT / A receptor interacts with BoNT / A to form a neurotoxin / receptor complex, and subsequently Refers to the ability of the complex to internalize into the cell cytoplasm.

  As used herein, the term “naturally occurring BoNT / A receptor” is generated from, but is not limited to, post-translational modifications, alternatively spliced transcripts, or spontaneous mutations. Refers to any BoNT / A receptor produced by naturally occurring processes, including the BoNT / A receptor isoform, and the BoNT / A receptor subtype. Naturally-occurring BoNT / A receptors include, but are not limited to, “Botulinum toxin screening assay” Ester Fernandez-Salas et al., US Patent Application Publication No. 2008/0003240; “Botulinum toxin screening assay” Ester Fernandez-Salas et al. "SV2 is a protein receptor for botulinum neurotoxin A", Min Dong et al., Science (2006); "Synaptic vesicle protein 2C is a phrenic nerve of botulinum neurotoxin A", US Patent Application Publication No. 2008/0182799; Mediate uptake into "S. Mahrhold et al, FEBS Lett. 580 (8): 2011-2014 (2006), each of which is incorporated herein by reference in its entirety. Blast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), synaptic vesicle sugar tamper Includes quality 2 (SV2) and GT1b-like complex ganglioside. Although not limited to naturally occurring FGFR2, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, or SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: : 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, for example, 1 or more, 2 or more, 3 or more, 4 or more, Substitution or deletion of 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more amino acids Lost or added Murrell. For example, one or more from SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, although not limited to naturally occurring FGFR3 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, Alternatively, a substitution, deletion or addition of 100 or more amino acids is included. Although not limited to naturally occurring SV2, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: : From 1 to 2, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 As mentioned above, the substitution, deletion or addition of 40 or more, 50 or more, or 100 or more amino acids is included.

  As used herein, the term “non-naturally occurring BoNT / A receptor variant” includes, but is not limited to, BoNT produced by genetic engineering using random mutagenesis or rational design. Refers to any BoNT / A receptor produced or engineered with the aid of human manual manipulation, including the / A receptor and the BoNT / A receptor produced by chemical synthesis. Non-limiting examples of non-naturally occurring BoNT / A variants include, for example, conserved BoNT / A receptor variants, non-conserved BoNT / A receptor variants, BoNT / A receptor chimeric variants and active BoNT / A receptor fragments. It is.

  As used herein, the term “non-naturally occurring BoNT / A receptor” includes, but is not limited to, BoNT / A produced by genetic engineering using random mutagenesis or rational design. It refers to any BoNT / A receptor whose structure has been modified with the aid of human manual manipulation, including the A receptor and the BoNT / A receptor produced by in vitro chemical synthesis. Non-limiting examples of non-naturally occurring BoNT / A receptors are eg “Botulinum toxin screening assay” Ester Fernandez-Salas et al., US Patent Application Publication No. 2008/0003240; “Botulinum toxin screening assay” Ester Fernandez-Salas et al., US Patent Application Publication No. 2008/0182799, each of which is incorporated herein by reference in its entirety. Non-naturally occurring BoNT / A receptors are SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, sequence. No: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or sequence Number: for example from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, from 70 More than 30, more than 40, more than 50, or more than 100 amino acids can be substituted, deleted or added.

  Thus, in an embodiment, the BoNT / A receptor is a naturally occurring BoNT / A receptor such as FGFR2, FGFR3 or SV2. In aspects of this embodiment, the BoNT / A receptor is a BoNT / A receptor isoform or a BoNT / A receptor subtype. In aspects of this embodiment, the naturally occurring BoNT / A receptor is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, The naturally occurring BoNT / A receptor of SEQ ID NO: 69 or SEQ ID NO: 70. In another aspect of this embodiment, the BoNT / A receptor comprises SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, Spontaneous BoNT / A acceptor having, for example, at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid identity with SEQ ID NO: 69 or SEQ ID NO: 70 Is the body.

  In another embodiment, the BoNT / A receptor is a non-naturally occurring BoNT / A receptor such as, for example, genetically engineered FGFR2, genetically engineered FGFR3, or genetically engineered SV2. In another aspect of this embodiment, the BoNT / A receptor comprises SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, A non-naturally occurring BoNT / A receptor having, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid identity with SEQ ID NO: 69 or SEQ ID NO: 70 . In another aspect of this embodiment, the BoNT / A receptor comprises SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, Relative to SEQ ID NO: 69 or SEQ ID NO: 70, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, A non-naturally occurring BoNT / A receptor having 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-adjacent amino acid substitutions, deletions or additions. In yet another aspect of this embodiment, the BoNT / A receptor is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. , SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 For example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more relative to SEQ ID NO: 69 or SEQ ID NO: 70 A non-naturally occurring BoNT / A receptor having 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more adjacent amino acid substitutions, deletions or additions.

  The BoNT / A receptor may be an endogenous BoNT / A receptor or an exogenous BoNT / A receptor. As used herein, the term “endogenous BoNT / A receptor” refers to an external source of BoNT / A receptor or BoNT / A because it is naturally encoded in the genome of the cell. Refers to the BoNT / A receptor naturally present in a cell such that the cell naturally expresses the BoNT / A receptor without the need for an external source of genetic material encoding the receptor. Expression of endogenous BoNT / A receptor may or may not be accompanied by environmental stimuli such as cell differentiation or promoter activation. For example, the following established cell lines express at least one endogenous BoNT / A receptor: BE (2) -M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108- 15, PC12, SH-SY5Y, SiMa and SK-N-BE (2) -C. The endogenous BoNT / A receptor may be only the naturally occurring BoNT / A receptor or a naturally occurring variant thereof.

  As used herein, the term “exogenous BoNT / A receptor” refers to an external source of a BoNT / A receptor or a genetic material that encodes a BoNT / A receptor by human manual manipulation. Refers to the BoNT / A receptor expressed in cells through the introduction of an external source. Expression of exogenous BoNT / A receptor may or may not be accompanied by environmental stimuli such as cell differentiation or promoter activation. As a non-limiting example, cells from an established cell line can be isolated by transient or stable transfection of a polynucleotide molecule encoding a BoNT / A receptor such as FGFR2, FGFR3 or SV2. More exogenous BoNT / A receptors can be expressed. As another non-limiting example, cells from an established cell line may contain one or more exogenous BoNT / A receptors, eg by protein transfection of a BoNT / A receptor such as FGFR2, FGFR3 or SV2. Can be expressed. The exogenous BoNT / A receptor may be a naturally occurring BoNT / A receptor or a naturally occurring variant thereof, or a non-naturally occurring BoNT / A receptor or a non-naturally occurring variant thereof.

  Thus, in an embodiment, cells from an established cell line express endogenous BoNT / A receptor. In aspects of this embodiment, the endogenous BoNT / A receptor expressed by cells from an established cell line is a naturally occurring BoNT / A receptor. In another aspect of this embodiment, the endogenous BoNT / A receptor expressed by cells from an established cell line is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, sequence SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 65 No: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70. In yet a further aspect of this embodiment, the endogenous BoNT / A receptor expressed by cells from an established cell line is a natural, eg, BoNT / A receptor isoform or BoNT / A receptor subtype. Developmental BoNT / A receptor. In another aspect of this embodiment, the endogenous BoNT / A receptor expressed by cells from an established cell line is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, for example at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90% or A naturally occurring BoNT / A receptor with at least 95% amino acid identity.

  In another embodiment, cells from established cell lines are engineered to transiently or stably express exogenous BoNT / A receptors. In aspects of this embodiment, cells from established cell lines are engineered to transiently or stably express the naturally occurring BoNT / A receptor. In other aspects of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or engineered to express the naturally occurring BoNT / A receptor of SEQ ID NO: 70. In yet another aspect of this embodiment, cells from established cell lines are transiently or stably generated naturally occurring BoNTs such as BoNT / A receptor isoforms or BoNT / A receptor subtypes. Engineered to express the / A receptor. In yet another aspect of this embodiment, cells from an established cell line are transiently or stably SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: : 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: : 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, for example at least 70% amino acid identity, at least 75%, at least 80%, at least 85%, at least 90% or at least 95 Engineered to express the naturally occurring BoNT / A receptor with% amino acid identity.

  In another aspect of the embodiment, cells from established cell lines are engineered to transiently or stably express non-naturally occurring BoNT / A receptors. In other aspects of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, for example at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid identity Engineered to express a non-naturally occurring BoNT / A receptor. In other aspects of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, Non-naturally occurring with 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-adjacent amino acid substitutions, deletions or additions Engineered to express the developmental BoNT / A receptor It is. In yet another aspect of this embodiment, cells from the established cell line are transiently or stably SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: : 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: : 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69 or SEQ ID NO: 70, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more non-naturally occurring amino acid substitutions, deletions or additions Manipulate to express the developmental BoNT / A receptor It is.

  In another embodiment, cells from an established cell line are engineered to transiently or stably express exogenous FGFR2, exogenous FGFR3, exogenous SV2, or any combination thereof. In aspects of this embodiment, cells from established cell lines are engineered to transiently or stably express naturally occurring FGFR2, naturally occurring FGFR3, naturally occurring SV2 or any combination thereof. In yet another aspect of this embodiment, cells from the established cell line transiently or stably express non-naturally occurring FGFR2, non-naturally occurring FGFR3, non-naturally occurring SV2 or any combination thereof To be manipulated. In yet another aspect of this embodiment, cells from an established cell line are transiently or stably generated naturally occurring FGFR2 or non-naturally occurring FGFR2, naturally occurring FGFR3 or nonnaturally occurring FGFR3, naturally occurring SV2 or Engineered to express non-naturally occurring SV2, or any combination thereof.

  Cells expressing one or more endogenous or exogenous BoNT / A receptors can be identified by routine methods including direct and indirect assays for toxin uptake. Assays that determine BoNT / A binding or uptake properties can be used to assess whether cells express the BoNT / A receptor. Such assays include, but are not limited to, cross-linking assays using labeled BoNT / A such as, for example, [125I] BoNT / A, [125I], for example, a variety of "Clostridial botulinum type C neurotoxins" Binding to neuroblastoma cell lines "Noriko Yokosawa et al., Infect. Immun. 57 (1): 272-277 (1989);" Botulinum Cl, D and E type neurotoxins to neuronal cell lines and synaptosomes " "Noriko Yokosawa et al. Toxicon 29 (2): 261-264 (1991); and" Identification of protein receptors for clostridial botulinum type B neurotoxins in rat brain synaptosomes "Tei-ichi Nishiki et al., J. Biol Chem. 269 (14): 10498-10503 (1994). Other non-limiting assays include immunocytochemical assays that use labeled or unlabeled antibodies to detect BoNT / A binding (eg “internalization of Clostridium botulinum type C precursor toxin into HT-29 cells). Receptors and transporters for "Atsushi Nishikawa et al., Biochem. Biophys. Res. Commun. 319 (2): 327-333 (2004)) and labeled or unlabeled antibodies to detect bound toxins Immunoprecipitation assays (see, eg, “Molecular Characterization of Clostridium Botulinum Type C Precursor Toxin Binding Subcomponents on Intestinal Epithelial Cells and Red Blood Cells” Yukako Fujinaga et al., Microbiology 150 (Pt 5): 1529-1538 (2004)) included. Antibodies useful in these assays include, but are not limited to, antibodies selected against BoNT / A, such as antibodies selected against BoNT / A receptors such as FGFR2, FGFR3 or SV2. And / or antibodies selected against gangliosides such as GD1a, GD1b, GD3, GQ1b or GT1b. If the antibody is labeled, cells or substrates after Western blot analysis, direct microscopic observation of antibody cell location, washing steps, flow cytometry, electrophoresis or capillary electrophoresis using techniques well known to those skilled in the art Molecular binding can be detected by various means including measurement of bound antibody. If the antibody is unlabeled, a labeled secondary antibody for indirect detection of the binding molecule can be used and detection can proceed as for the labeled antibody. It will be appreciated that these and similar assays that determine BoNT / A uptake properties or characteristics may be useful in identifying cells that express endogenous or exogenous or BoNT / A receptors.

  Assays that monitor the release of molecules following exposure to BoNT / A can also be used to assess whether cells express one or more endogenous or exogenous BoNT / A receptors. . In these assays, after release of BoNT / A, inhibition of release of the molecule will occur in cells expressing the BoNT / A receptor. Well-known assays measure the inhibition of radiolabeled catecholamine release, such as [3H] noradrinaline or [3H] dopamine release from neurons (eg “calcium dependence less sensitive to tetanus toxin and botulinum toxin type F) Evidence for Sexual Vesicle Transmitter Release ”A Fassio et al., Neuroscience 90 (3): 893-902 (1999); and“ Catecholamine release sensitivity to botulinum toxins C1 and E makes it a readily releasable pool. "Sara Stigliani et al., J. Neurochem. 85 (2): 409-421 (2003)) or fluorometric procedures are used to measure catecholamine release. (For example, a botulinum neurotoxin represented by a toxin derivative that binds to ecto-acceptors and blocks intracellular transmitter release. The role of interchain disulfides or their associated thiols in A internalization "Anton de Paiva et al., J. Biol. Chem. 268 (28): 20838-20844 (1993);" 25 kDa with botulinum toxin A or B Explicit exocytosis response of intact and permeabilized chromaffin cells after cleavage of synaptosome-related protein (SNAP-25) or synaptobrevin "Gary W. Lawrence et al., Eur. J. Biochem. 236 ( 3): 877-886 (1996); and "Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: Correlation with its blockade of catecholamine release" Patrick Foran et al., Biochemistry 35 (8): 2630-2636 (1996)). Other non-limiting examples include assays that measure inhibition of hormone release from endocrine cells such as, for example, anterior pituitary cells or ovarian cells. It is understood that these and similar assays for molecular release may be useful for identifying cells that express endogenous or exogenous or BoNT / A receptors.

  Assessing whether a cell expresses one or more endogenous or exogenous BoNT / A receptors using an assay that detects cleavage of the BoNT / A substrate following exposure to BoNT / A You can also. In these assays, after BoNT / A treatment, BoNT / A substrate cleavage product production or loss of intact BoNT / A substrate is detected in cells expressing the BoNT / A receptor. Non-limiting examples of specific western blot analysis and well-characterized reagents, conditions and protocols include, but are not limited to, Amersham Biosciences, Piscataway, NJ; Bio-Rad Laboratories, Hercules, CA; Pierce Biotechnology, Inc., Rockford, IL; readily available from commercial manufacturers including Promega Corporation, Madison, WI; and Stratagene, Inc., La Jolla, CA. These and similar assays for BoNT / A substrate cleavage may be useful for identifying cells that express endogenous or exogenous BoNT / A receptors.

  Non-limiting examples include assaying for BoNT / A incorporation using Western blot analysis using BoNT / A SNAP-25 cleavage products or antibodies that recognize both cleaved and uncleaved forms of SNAP-25. it can. Examples of α-SNAP-25 antibodies useful in these assays include, but are not limited to, SMI-81α-SNAP-25 mouse monoclonal antibody (Sternberger Monoclonals Inc., Lutherville, MD), CI 71.1 mouse α. -SNAP-25 monoclonal antibody (Synaptic Systems, Goettingen, Germany), CI71.2α-SNAP-25 mouse monoclonal antibody (Synaptic Systems, Goettingen, Germany), SP12α-SNAP-25 mouse monoclonal antibody (Abcam, Cambridge, MA), α-SNAP-25 rabbit polyclonal antiserum (Synaptic Systems, Goettingen, Germany), α-SNAP-25 rabbit polyclonal antiserum S9684 (Sigma, St Louis, MO) and α-SNAP-25 rabbit polyclonal antiserum (Abcam, Cambridge) , MA).

  Aspects of the present disclosure provide cells made to express exogenous SNAP-25 and / or one or more exogenous BoNT / A receptors via manual or recombinant manipulation. . Cells useful for expressing exogenous SNAP-25 and / or one or more exogenous BoNT / A receptors via manual or recombinant manipulation include endogenous SNAP-25 and / or Also included are neuronal cells and non-neuronal cells that may or may not express one or more endogenous BoNT / A receptors. Such genetically engineered or recombinantly engineered cells are exogenous SNAP-25 and one or more exogenous under the control of constitutive, tissue specific, cell specific or inducible promoter elements, enhancer elements or both. It is further understood that the BoNT / A receptor can be expressed. Any cell is useful as long as the cell can be genetically engineered or recombinantly engineered to express exogenous SNAP-25 and / or one or more exogenous BoNT / A receptors, It is understood that BoNT / A poisoning can occur.

Exogenous encoding the components necessary for the cell so that BoNT / A performs an overall cellular mechanism by which proteolysis cleaves a SNAP-25 substrate such as SNAP-25, FGFR2, FGFR3 or SV2. Useful methods for introducing a polynucleotide molecule into a cell include, but are not limited to, for example, calcium phosphate mediated, diethyl-aminoethyl (DEAE) dextran mediated, lipid mediated, polyethyleneimine (PEI) mediated, polylysine mediated and Chemical mediated distribution methods such as polybrene mediated; physics mediated methods such as biolistic particle distribution, microinjection, protoplast fusion and electroporation; and such as retrovirus mediated transfection Include virus-mediated distribution method, for example, "introduction into cloned genes cultured mammalian cells" Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3 rd ed. Pp. 16.1-16.62 (2001); "Transfer and expression of foreign genes in mammalian cells" Alessia Colosimo et al., Biotechniques 29 (2): 314-318, 320-322, 324 (2000); "For gene transfer into neurons Technology "Philip Washbourne & A. Kimberley McAllister, Philip Washbourne & A. Kimberley McAllister, Curr. Opin. Neurobiol. 12 (5): 566-573 (2002); and Current Protocols in Molecular Biology, John Wiley and Sons, pp 9.16. .4-9.16.11 (2000), each of which is incorporated herein by reference in its entirety. The selection of a specific method for introducing the polynucleotide molecule into the cell is in part necessary for the cell to perform the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis. One skilled in the art will appreciate that the component depends on whether the cell contains transiently or stably. A non-limiting example of a polynucleotide molecule that encodes the components necessary for a cell to effect the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis is: SEQ ID NO: : 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137 or FGFR2 polynucleotide molecule of SEQ ID NO: 138; SEQ ID NO: 139, SEQ ID NO: 140 or FGFR3 polynucleotide molecule of SEQ ID NO: 141; SEQ ID NO: 142, SEQ ID NO: 143 or SV2 polynucleotide molecule of SEQ ID NO: 144; and SNAP of SEQ ID NO: 145 or SEQ ID NO: 146 -25 polynucleotide molecules.

  Chemical mediated distribution methods are well known to those skilled in the art and are described, for example, in “Adhesion of calcium phosphate and transfection of suspension cells” Martin Jordan & Florian Worm, Methods 33 (2): 136-143 (2004); Polyethyleneimine Project for the Distribution of Plasmids "in Chun Zhang et al., Methods 33 (2): 144-150 (2004), each of which is incorporated herein by reference in its entirety. The Such chemical-mediated partitioning methods can be prepared by standard procedures and are commercially available, eg, CellPhect transfection kit (Amersham Biosciences, Piscataway, NJ); mammalian transfection kits, calcium phosphate and DEAE dextran ( Stratagene, Inc., La Jolla, CA); Lipofectamine ™ transfection reagent (Invitrogen, Inc., Carlsbad, CA); ExGen 500 transfection kit (Fermentas, Inc., Hanover, MD) and SuperFect and Effectene transfection See kit (Qiagen, Inc., Valencia, CA).

  Physics-mediated methods are well known to those skilled in the art and, for example, “Plasmid-mediated gene transfer in neurons using biolistic technology” Jeike E. Biewenga et al., J. Neurosci. Methods. 71 (1): 67-75. (1997); “Biolistic and Geolistic Transfection: Distributing DNA and lipophilic dyes to mammalian cells using a gene gun” John O'Brien & Sarah CR Lummis, Methods 33 (2): 121- 125 (2004); “in vitro and in vivo electric field mediated permeabilization, gene transfer and expression” M. Golzio et al., Methods 33 (2): 126-135 (2004); and “for gene transfer into primary cells. New non-viral methods "Oliver Greschet al., Methods 33 (2): 151-163 (2004), each of which is hereby incorporated by reference in its entirety.

  Virus-mediated partitioning methods are well known to those skilled in the art and are described, for example, in “Adenoviruses and adeno-associated virus vectors” Chooi M. Lai et al., DNA Cell Biol. 21 (12): 895-913 (2002); Basal expression vectors: planning and application "Ilya Frolov et al., Proc. Natl. Acad. Sci. USA 93 (21): 11371-11377 (1996);" Lentiviral vectors for the distribution of DNA to mammals " Roland Wolkowicz et al., Methods Mol. Biol. 246: 391-411 (2004); “Baculovirus vector: a novel mammalian cell gene distribution vehicle and its application” A. Huser & C. Hofmann, Am. J. Pharmacogenomics 3 (1): 53-63 (2003); “Transient generation of retroviral and lentiviral based vectors for transduction of mammalian cells” Tiziana Tonini et al., Methods Mol. Biol. 285: 141-148 (2004); “Tetracycline response promo Strict control of gene expression in eukaryotic cells by a coagulator ”Manfred Gossen and Hermann Bujard, US Pat. No. 5,464,758;“ Methods for modulating gene expression ”Hermann Bujard and Manfred Gossen, US Pat. No. 5,814,618; Polynucleotides encoding hormone receptor polypeptides and cells transformed with the same "David S. Hogness, US Pat. No. 5,514,578;" Polynucleotides encoding insect ecdysone receptors "David S. Hogness, US Pat. “Progesterone receptor with C-terminal hormone binding domain truncation” Elisabetta Vegeto et al., US Pat. No. 5,364,791; “Mutated steroid hormone receptor, methods for its use and molecular switch for gene therapy” Elisabetta Vegeto et al. It described in No. 5874534 (the each which is hereby incorporated by reference in its entirety). Such virus-mediated partitioning methods can be prepared by standard procedures and are commercially available, eg, ViraPower ™ adenovirus expression system (Invitrogen, Inc., Carlsbad, CA) and ViraPower ™ adeno Virus Expression System Description Manual 25-0543 Version A, Invitrogen, Inc. (June 15, 2002); and AdEasy ™ Adenoviral Vector System (Stratagene, Inc., La Jolla, CA) and AdEasy ™ Adeno See Viral Vector System Description Manual 0640004f (Stratagene, Inc.). In addition, such virus-mediated distribution systems can be prepared by standard methods and are commercially available, for example, BD ™ Tet-Off and Tet-On gene expression systems (BD Biosciences-Clonetech, Palo Alto, CA) and BD ™ Tet-Off and Tet-On gene expression system user manual PT3001-1 (BD Biosciences Clonetech) (March 14, 2003), GeneSwitch ™ system (Invitrogen, Inc., Carlsbad, CA) ) And GeneSwitch ™ system A mifepristone regulated expression system version D, 25-0313 (Invitrogen, Inc.) (November 4, 2002) for mammalian cells; ViraPower ™ lentiviral expression system ( Invitrogen, Inc., Carlsbad, CA) and ViraPower ™ Lentiviral Expression System Description Manual 25-0501 Version E (Invitrogen, Inc.) (December 8, 2003); Want Complete Control ® Retroviral derived mammalian expression system (Stratagene, La Jolla, CA) and Complete Control by reference (registered trademark) retrovirus derived mammalian expression systems described manual 064005e on.

  Thus, in an embodiment, cells from an established cell line that are highly sensitive to BoNT / A intoxication are transferred to the cell to perform the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis. It contains transiently the polynucleotide molecules encoding the necessary components. In another embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication are used to perform the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis. A polynucleotide molecule encoding a plurality of components necessary for the above is transiently contained. In aspects of this embodiment, cells from an established cell line that are highly sensitive to BoNT / A intoxication transiently contain polynucleotide molecules encoding FGFR2, FGFR3, SV2 or SNAP-25. In aspects of this embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication are SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, Transiently contains a polynucleotide molecule encoding FGFR2 of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137 or SEQ ID NO: 138. In another aspect of this embodiment, cells from an established cell line that are highly sensitive to BoNT / A intoxication are polynucleotide molecules encoding FGFR3 of SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141. Contains transiently. In yet another aspect of this embodiment, cells from an established cell line that are sensitive to BoNT / A poisoning are polynucleotide molecules encoding SV2 of SEQ ID NO: 142, SEQ ID NO: 143 or SEQ ID NO: 144. Is included transiently. In yet another aspect of this embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication transiently transduce a polynucleotide molecule encoding SNAP-25 of SEQ ID NO: 145 or SEQ ID NO: 146. Contains to sex.

  In another embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication are used to perform the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis. It stably contains a polynucleotide molecule that encodes a component necessary for the preparation. In another embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication are used to perform the overall cellular mechanism by which BoNT / A cleaves the SNAP-25 substrate by proteolysis. It stably contains a polynucleotide molecule that encodes a plurality of constituent elements necessary for. In aspects of this embodiment, cells from an established cell line that are highly sensitive to BoNT / A intoxication stably contain a polynucleotide molecule encoding FGFR2, FGFR3, SV2 or SNAP-25. In aspects of this embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication are SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, It stably contains a polynucleotide molecule encoding FGFR2 of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137 or SEQ ID NO: 138. In another aspect of this embodiment, cells from an established cell line sensitive to BoNT / A intoxication stabilize the polynucleotide molecule encoding FGFR3 of SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141. Contained. In yet another aspect of this embodiment, cells from an established cell line that are sensitive to BoNT / A poisoning are polynucleotide molecules encoding SV2 of SEQ ID NO: 142, SEQ ID NO: 143 or SEQ ID NO: 144. Is stably contained. In yet another aspect of this embodiment, cells from an established cell line that are sensitive to BoNT / A intoxication stabilize the polynucleotide molecule encoding SNAP-25 of SEQ ID NO: 145 or SEQ ID NO: 146. Contained.

  As mentioned above, it provides the overall cellular mechanism by which BoNT / A cleaves SNAP-25 substrates such as SNAP-25, FGFR2, FGFR3 or SV2 disclosed herein by proteolysis. The exogenous components necessary for the cells can be introduced into the cells. Any and all methods useful for introducing such exogenous components into a cell population with a partitioning agent are those exogenous that the method is disclosed herein in at least 50% of the cells within a given cell population. It may be useful on the condition that the component is introduced transiently. Thus, aspects of this embodiment are disclosed herein, for example, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of a given cell population, whereby BoNT / A is proteolytically disclosed, for example, herein. Cell populations that transiently contain the exogenous components necessary for the cell to perform the overall cellular mechanism of cleaving a SNAP-25 substrate, such as SNAP-25, FGFR2, FGFR3 or SV2. As used herein, the term “partition agent” refers to the interior of a polypeptide covalently linked, non-covalently linked, or in any other manner associated with a cell. Refers to any molecule that allows or enhances translocation. Thus, the term “partitioning agent” includes, but is not limited to protein. Peptides, peptide mimetics, small molecules, polynucleotide molecules, liposomes, lipids, viruses, retroviruses and, but not limited to, covalently or non-covalently linked molecules include cell membranes, cell cytoplasm or nucleus Includes cells that transport to The term “partition agent” further encompasses molecules that are internalized by any mechanism, including those that function via receptor-mediated endocytosis and those that are independent of receptor-mediated endocytosis. Understood.

  Partitioning agents are agents that allow or enhance cellular uptake of covalently linked FGFR2, FGFR3, SV2 or SNAP-25-like components, such as by chemical conjugation or genetically generated fusion proteins But you can. Methods for covalently linking to partitioning agents and methods for using such agents are described, for example, in “Protein Transduction Systems and Methods of Use”, Steven F. Dowdy, International Publication No. WO 00/34308; “Intracellular addressing of active molecules” Peptides that can be used as vectors "Gerard Chassaing and Alain Prochiantz, US Pat. No. 6,080,724;“ Fusion proteins comprising a TAT-derived transport moiety ”Alan Frankel et al., US Pat. No. 5,674,980;“ TAT-derived transport ” "Polypeptide conjugates" Alan Frankel et al., US Pat. No. 5,747,641; “TAT-derived transport polypeptides and fusion proteins” Alan Frankel et al., US Pat. No. 5,804,604; “Use of transport proteins” Peter FJ O'Hare et al., US Patent No. 6734167; “For the transfer of biologically active molecules into cells Method "Yao-Zhong Lin and Jack J. Hawiger, US Pat. No. 5,807,746;" Method for transfer of biologically active molecules into cells "Yao-Zhong Lin and Jack J. Hawiger, US Patent 6043339; "Sequences and methods for genetic manipulation of proteins with cell membrane translocation activity" Yao-Zhong Lin et al., US Pat. No. 6,248,558; "Sequences for genetic manipulation of proteins with cell membrane translocation activity and Methods "Yao-Zhong Lin et al., US Pat. No. 6,432,680;" Methods for Transfer of Biologically Active Molecules into Cells "Jack J. Hawiger et al., US Pat. No. 6,495,518; Sequences and methods for genetic manipulation of proteins having "Yao-Zhong Lin et al., US Pat. No. 6,780,843;" Methods and compositions for enhancing transport across biological membranes "Jona than B. Rothbard and Paul A Wender, US Pat. No. 6,306,993; “Methods and compositions for enhancing transport across biological membranes” Jonathan B. Rothbard and Paul A Wender, US Pat. No. 6,495,663; Fusion proteins for protein partitioning ", Pamela B. Davis et al., US Pat. No. 6,287,817, each of which is incorporated herein by reference in its entirety.

  The partitioning agent may be an agent that enables or enhances cellular uptake of non-covalently associated components such as FGFR2, FGFR3, SV2 or SNAP-25. Methods that function in the absence of covalent linkages and methods of using such agents are described, for example, in “Peptide-Mediated Transfection Agents and Methods of Use” Gilles Divita et al., US Pat. No. 6,841,535; “Intracellular protein partitioning compositions and uses of Methods "Philip L Felgner and Olivier Zelphati, US 2003/0008813; and" Intracellular partitioning of small molecules, proteins and nucleic acids "Michael Karas, US 2004/029797 (each of which is all in its entirety) Which is incorporated herein by reference). Such peptide partitioning agents can be prepared and used by standard methods and are commercially available, eg, CHARIOT ™ reagent (Active Motif, Carlsbad, CA); BIO-PORTER ™ reagent ( Gene Therapy Systems, Inc., San Diego, CA), BIO TREK ™ protein partitioning reagent (Stratagene, La Jolla, CA) and PRO-JECT ™ protein transfection reagent (Pierce Biotechnology Inc., Rockford, IL) Please refer to.

Aspects of the present disclosure comprise in part a sample comprising BoNT / A. As used herein, the term “sample comprising BoNT / A” refers to any biological material that contains or potentially contains active BoNT / A. Without limitation, purified, partially purified or unpurified BoNT / A; recombinant single- or double-chain toxins with natural or non-naturally occurring sequences; modified protease specificity Recombinant BoNT / A with recombinant BoNT / A with altered cell specificity; Bulk BoNT / A; eg BOTOX®, DYSPORT® / RELOXIN®, XEOMIN® , PURTOX (registered ^trademark), NEURONOX (registered trademark), formulated BoNT / a product containing BTX-a, and; such as bacteria, yeast, insect or cells, or crude from mammalian sources, fractionated or Partially purified cell lysate; blood, plasma or serum; raw, cooked, partially cooked or processed food; beverage; animal feed; soil sample; water sample; Various samples including quality; lotion; cosmetics; as well as clinical formulations can be assayed according to the methods disclosed herein. The term sample is understood to include, but is not limited to, tissue samples including mammalian tissue samples, livestock tissue samples such as sheep, cattle and pig tissue samples; primate tissue samples; and human tissue samples. The Such samples include, but are not limited to, intestinal samples such as infant bowel samples, and tissue samples obtained from wounds. By way of non-limiting example, a method for detecting picomolar amounts of BoNT / A activity is used to determine the presence or activity of BoNT / A in a food or beverage sample; for example, exposed to BoNT / A or 1 For assaying samples from humans or animals with one or more symptoms of botulism; to track activity during the production and purification of bulk BoNT / A; for use in pharmaceutical or cosmetic applications To assay the formulated BoNT / A product; or to assay the serum of a subject for the presence or absence of α-BoNT / A neutralizing antibodies;

  Thus, in an embodiment, a sample comprising BoNT / A is a sample comprising any amount of BoNT / A. In aspects of this embodiment, the sample comprising BoNT / A comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, about 100 pg or less, about 10 pg or less, or about 1 pg or less BoNT / A. In other aspects of this embodiment, the sample comprising BoNT / A is about 1 μM or less, about 100 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM or less, about 1 pM or less, about 100 fM or less, About 10 fM or less or about 1 fM or less of BoNT / A.

Aspects of the present disclosure from in some treated cells, isolating a SNAP-25 component comprising a SNAP-25 having a carboxyl-terminus at the P ₁ residue of the BoNT / A cleavage site scissile bond Comprising. The term "BoNT / A comprising a SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the cleavage site scissile bond SNAP-25 component" as used herein, SNAP-25 cleavage Refers to the cellular component containing the product. Any suitable method for enriching or isolating SNAP-25 components is useful, including but not limited to cell lysis protocols, spin column purification protocols, immunoprecipitation, affinity purification and protein chromatography. It is assumed that this is possible.

  Aspects of the disclosure comprise in part an α-SNAP-25 antibody linked to a solid support. As used herein, the term “solid support” is synonymous with “solid phase” and is used for immobilization of the α-SNAP-25 antibody disclosed herein. Refers to any matrix that can. Non-limiting examples of solid supports include, for example, tubes; plates; columns; pins or “dipsticks”; magnetic particles, beads, or other spherical or fibrous chromatography such as agarose, sepharose, silica and plastics. Medium; and sheets or membranes such as, for example, nitrocellulose and polyvinylidene fluoride (PVDF). Various materials such as glass, carbon, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, nylon, diazocellulose, or starch can be used to construct the solid support. The selected solid support can have physical properties that allow it to be easily separated from soluble or unbound material and generally remove unbound material such as excess reagents, reaction byproducts or solvents ( Allowing separation or otherwise removal from the solid support binding assay component (eg, by washing, filtration, centrifugation, etc.). Non-limiting examples of how to make and use solid supports include, for example, Molecular Cloning, A Laboratory Manual, supra, (2001); and Current Protocols in Molecular Biology, supra, (2004) (each of which is cited in its entirety) As a part of this specification).

Aspects of the present disclosure selectively bind to alpha-SNAP-25 antibody and the BoNT / A cleavage site cleavable in part on SNAP-25 epitope having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond antibodies comprising SNAP-25 cleavage product having a carboxyl-terminus at the P ₁ residue of the coupling - comprises detecting the presence of the antigen complex. This disclosed immune basis using any detection system provided that the signal to noise ratio can distinguish signals from antibody-antigen complexes from background signals to a statistically significant extent. It is envisioned that the method aspects of can be implemented. Non-limiting examples of immuno-based detection systems include Western blotting and dot blotting-like immunoblot analysis, immunoprecipitation analysis, enzyme-linked immunosorbent analysis (ELISA) and sandwich ELISA. Autoradiography (AU) with imaging or phosphor imaging, chemiluminescence (CL), electrochemiluminescence (ECL), bioluminescence (BL), fluorescence, resonance energy transfer, plane polarization, colorimetric or flow cytometry (FC) can be used to achieve signal detection. Descriptions of immune-based detection systems include, for example, “chemiluminescence” Michael M. Rauhut, Kirk-Othmer Concise Encyclopedia of Chemical Technology (Ed. Grayson, 3rd ed, John Wiley and Sons, 1985); "A review of recent trends" AW Knight, Trends Anal. Chem. 18 (1): 47-62 (1999); "Recent applications of electrochemiluminescence in chemical analysis" KAFahnrich et al. Talanta 54 (4): 531 -559 (2001);.... "commonly used techniques in molecular cloning," pp A8.1-A8-55 (Sambrook & Russell , eds, molecular cloning A Laboratory Manual, Vol 3, 3 rd ed 2001 );. "detection system" pp A9.1-A9-49 (Sambrook & Russell , eds, Molecular Cloning A Laboratory Manual, Vol 3, 3 rd ed 2001);.... Electrogenerated Chemiluminescence (Ed Allen J. Bard, Marcel Dekker, Inc., 2004), each of which is incorporated herein by reference in its entirety.

  Sandwich ELISA (or sandwich immunoassay) is a method based on two antibodies that bind to different epitopes on an antigen. Capture antibodies with high binding specificity for the antigen of interest are bound to the solid surface. The antigen is then added, followed by a second antibody called the detection antibody. The detection antibody binds the antigen to a different epitope than the capture antibody. The antigen is therefore “sandwiched” between the two antibodies. Antibody binding affinity for antigen is usually the main determinant of immunoassay sensitivity. As the antigen concentration increases, the amount of detection antibody increases and the response measured is higher. In order to quantify the degree of binding, different reporter systems can be used, such as an enzyme attached to a secondary antibody and a reporter substrate, where the enzyme reaction is read out as a detection signal. The signal produced is proportional to the amount of target antigen present in the sample. The reporter substrate used to measure the binding event determines the detection format. A spectrophotometric plate reader is used for colorimetric detection. In addition, chemiluminescent and electrochemiluminescent substrates have been developed that can amplify the signal and read with a luminescent reader. The reporter can also be a fluorescent readout, where the enzyme step of the assay is replaced with a fluorophore, and the readout is then measured using a fluorescent reader. Reagents and protocols necessary to perform ECL sandwich ELISA including MSD sandwich ELISA-ECL detection platform (Meso Scale Discovery, Gaithersburg, MD) without exception are commercially available.

Thus, in an embodiment, an α-SNAP-25 antibody that selectively binds to a SNAP-25 epitope having a carboxyl terminus at the P ₁ residue of a BoNT / A cleavage site cleavable linkage and a P of a BoNT / A cleavage site cleavable linkage. Detection of the presence of an antibody-antigen complex comprising a SNAP-25 cleavage product with a carboxyl terminus at _one residue can be performed using immunoblot analysis, immunoprecipitation analysis, ELISA or sandwich ELISA. . In aspects of this embodiment, AU, CL, ECL or BL immunoblot analysis, AU, CL, ECL, BL or FC immunoprecipitation analysis, AU, CL, ECL, BL or FC ELISA, or AU, CL, ECL, BL Alternatively, detection is performed using FC sandwich ELISA.

Aspects of the present disclosure can be implemented in a singleplex or multiplex fashion. Immuno-based method of detecting BoNT / A activity performed in a single plex manner, SNAP-25 cleavage product having a carboxyl-terminus at the _{P 1} residue of the alpha-SNAP-25 antibody and the BoNT / A cleavage site scissile bond Only the presence of the antibody-antigen complex comprising the product is detected. An immuno-based method of detecting BoNT / A activity performed in a multiplex fashion is to detect the presence of one or more antibody-antigen complexes simultaneously; one of which is α- SNAP-25 antibody and the BoNT / a cleavage site scissile bond _{P 1} residue comprising SNAP-25 cleavage product having a carboxyl-terminus at the antibody - an antigen complex; and (s) other things Antibody-antigen complexes against different proteins such as second, third, fourth, etc. For example, sample to sample variability by normalizing the amount of α-SNAP-25 / SNAP-25 antibody-antigen complex detected relative to the amount of antibody-antigen complex detected for the second protein. A second protein can be used as an internal control to minimize As such, the second protein is one that is consistently expressed by the cell, usually a housekeeping protein. Non-limiting examples of useful second proteins include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), syntaxin, cytokines. Methods for performing immune-based assays in a multiplex format are described, for example, in “microarray format multiplex sandwich assays” UB Nielsen and BH Geierstanger, J. Immunol. Methods. 290 (1-2): 107-120 (2004); Quantitative protein profiling using antibody arrays "R. Barry and M, Soloviev, Proteomics, 4 (12): 3717-3726 (2004);" Immunoassay using multiplex molecular diagnostics and emerging microarray technology "MM Ling et al , Expert Rev Mol Diagn. 7 (1): 87-98 (2007); "ELISA and multiplex technology for cytokine measurements in inflammation and aging studies" SX Leng et al., J Gerontol A Biol Sci Med Sci 63 (8): 879-884 (2008), each of which is hereby incorporated by reference in its entirety.

Thus in one embodiment, SNAP-25 cleavage product having a carboxyl-terminus at the _{P 1} residue of the BoNT / the immuno-based method of detecting A activity, α-SNAP-25 antibody and the BoNT / A cleavage site scissile bond Run in a single plex fashion by detecting only the presence of antibody-antigen complexes comprising the product. In another embodiment, BoNT / the immuno-based method of detecting A activity, α-SNAP-25 SNAP- 25 cleavage product having a carboxyl-terminus at the _{P 1} residue of the antibody and the BoNT / A cleavage site scissile bond And performed in a multiplex fashion by simultaneously detecting the presence of an antibody-antigen complex comprising at least one other antibody-antigen complex against a protein other than SNAP-25, eg, GAPDH or syntaxin To do.

  Aspects of the present disclosure provide, in part, a method for determining BoNT / A immune resistance. As used herein, the term “BoNT / A immune resistance” is used to reduce the effectiveness of a treatment, whether the mammal's immune response is direct or indirect. It means a mammal that does not respond well to A treatment or that exhibits a reduced beneficial effect of BoNT / A treatment. A non-limiting example of reduced efficacy would be the presence in mammals of at least one α-BoNT / A neutralizing antibody that binds to BoNT / A toxin in a manner that reduces or protects the specificity or activity of the toxin. . As used herein, the term “BoNT / A treatment” refers to a treatment, therapy, cure, offsetting something undesirable in a mammal in need of neuromodulation using BoNT / A toxin. BoNT / with medical, therapeutic, curative, cosmetic, therapeutic or any other beneficial effect of recovery, rehabilitation or any other means, or one or more controlled doses Meaning administration of a pharmaceutical, preparation or mixture of A toxin to a mammal. BoNT / A treatment includes, but is not limited to, the use of any naturally occurring or modified fragment thereof in any formulation combined with any carrier or active ingredient and administered by any route of administration. Illustratively, the well-known BoNT / A treatment is BOTOX® treatment.

Aspects of the present disclosure provide, in part, a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibody. As used herein, the term “test sample” refers to any biological material that contains or potentially contains at least one α-BoNT / A antibody. The α-BoNT / A antibody may be an anti-BoNT / A neutralizing antibody or an anti-BoNT / A non-neutralizing antibody. As used herein, the term “anti-BoNT / A neutralizing antibody” refers to BoNT in such a manner as to reduce or prevent the toxin from exerting its effect in BoNT / A treatment under physiological conditions. / A refers to any α-BoNT / A antibody that will bind to the toxin region. As used herein, the term “α-BoNT / A non-neutralizing antibody” binds to a region of BoNT / A toxin under physiological conditions, but the toxin is effective in treating BoNT / A. Means any α-BoNT / A antibody that will not protect against. It is envisioned that any and all samples that can contain α-BoNT / A antibodies, including but not limited to blood, plasma, serum and lymph, can be used in this method. In addition, α-BoNT / A antibodies against BoNT / A toxins include, but are not limited to, birds and mammals including mice, rats, goats, sheep, horses, donkeys, cattle, primates and humans. Any and all organisms that can be raised can serve as a source for the sample. Non-limiting examples of specific protocols for blood collection and serum preparation include, for example, Marjorie Schaub Di Lorenzo & Susan King Strasinger, BLOOD COLLECTION IN HEALTHCARE (FA Davis Company, 2001); and Diana Garza & Kathleen Becan-McBride, PHLEBOTOMY HANDBOOK: (. Prentice Hall, 6 th ed, 2002) BLOOD COLLECTION ESSENTIALS are described. These protocols are routine procedures and are well within the purview of those skilled in the art and from the teachings herein. Before exposure to BoNT / A toxin, after a single BoNT / A treatment, after multiple BoNT / A toxin treatments, before onset of resistance to BoNT / A therapy, or resistance to BoNT / A therapy A test sample can be obtained from the organism after the onset of.

  Aspects of the present disclosure provide, in part, a control sample. As used herein, the term “control sample” refers to any sample in which the presence or absence of a test sample is known, and includes both negative and positive control samples. Negative control samples for neutralization of α-BoNT / A antibody can be obtained from individuals who have never been exposed to BoNT / A and include, but are not limited to, the same individual supplying the test sample Samples taken from before the BoNT / A treatment; samples taken from different individuals who have never been exposed to BoNT / A; multiples never exposed to BoNT / A Pooled samples taken from different individuals. Positive control samples for α-BoNT / A neutralizing antibodies can be obtained from individuals who manifest BoNT / A immune resistance and are positive when tested in a patient-based test assay, without limitation. Individuals that are positive in testing in an in vivo bioassay; and individuals that are hyperimmune, such as BoNT / A vaccinated individuals.

It is further foreseen that the α-BoNT / A antibody can be purified from the sample. The anti-BoNT / A antibody can be purified from the sample using a variety of procedures including, but not limited to, protein A / G chromatography and affinity chromatography. Non-limiting examples of specific protocols for purifying antibodies from a sample, for example ANTIBODIES: A LABORATORY MANUAL; USING ANTIBODIES (Edward Harlow & David Lane, eds, Cold Spring Harbor Laboratory Press, 2 nd ed 1998..): A LABORATORY MANUAL: PORABLE PROTOCOL NO I (Edward Harlow & David Lane, Cold Spring Harbor Laboratory Press, 1998); and MOLECULAR CLONING, A LABORATORY MANUAL, supra, (2001), which is incorporated herein by reference. be written. In addition, non-limiting examples of antibody purification methods and well-characterized reagents, conditions and protocols include, but are not limited to, Pierce Biotechnology, Inc., Rockford, IL; and Zymed Laboratories, Inc., South San It is readily available from commercial manufacturers including Francisco, CA. These protocols are routine procedures that are well within the skill of the art.

  Thus, in an embodiment, the sample comprises blood. In aspects of this embodiment, the sample comprises mouse blood, rat blood, goat blood, sheep blood, horse blood, donkey blood, bovine blood, primate blood or human blood. In another embodiment, the sample comprises plasma. In aspects of this embodiment, the test sample comprises mouse plasma, rat plasma, goat plasma, sheep plasma, horse plasma, donkey plasma, bovine plasma, primate plasma or human plasma. In another embodiment, the sample comprises serum. In aspects of this embodiment, the sample comprises mouse serum, rat serum, goat serum, sheep serum, horse serum, donkey serum, bovine serum, primate serum and human serum. In another embodiment, the sample comprises lymph fluid. In aspects of this embodiment, the sample comprises mouse lymph, rat lymph, goat lymph, sheep lymph, horse lymph, donkey lymph, bovine lymph, primate lymph or human lymph. In yet another embodiment, the sample is a test sample. In yet another embodiment, the sample is a control sample. In aspects of this embodiment, the control sample is a negative control sample or a positive control sample.

Aspects of the present disclosure in some detected the amount of SNAP-25 having a carboxyl-terminus at _{the P 1} residue of the detected BoNT / A cleavage site scissile bond in step (d) in step (e) BoNT / It provides for comparing the amount of SNAP-25 having a carboxyl-terminus at the P ₁ residue of the a cleavage site scissile bond. In an embodiment, the amount of SNAP-25 cleavage product in the test sample is high compared to the amount of SNAP-25 cleavage product in the control sample. In aspects of this embodiment, an increased amount of SNAP-25 cleavage product in the test sample compared to the positive control sample indicates a reduction or lack of BoNT / A immunoresistance in the mammal. In another aspect of this embodiment, an equal amount of SNAP-25 cleavage product in the test sample compared to the negative control sample indicates a reduction or lack of BoNT / A immunoresistance in the mammal. . In another embodiment, the amount of SNAP-25 cleavage product in the test sample is low compared to the amount of SNAP-25 cleavage product in the control sample. In aspects of this embodiment, a reduced or equivalent amount of SNAP-25 cleavage product in the test sample compared to the positive control sample indicates an increase or presence of BoNT / A immunoresistance in the mammal. Indicates. In another aspect of this embodiment, a lower amount of SNAP-25 cleavage product in the test sample compared to the negative control sample indicates an increase or presence of BoNT / A immunoresistance in the mammal.

  Any and all assay conditions suitable for detecting the presence of α-BoNT / A neutralizing antibodies in a sample, such as linear and non-linear assay conditions, are disclosed herein. Useful in the method. In an embodiment, the assay conditions are linear. In aspects of this embodiment, the assay amount of BoNT / A is in excess. In another aspect of this embodiment, the assay amount of BoNT / A is rate limiting. In another aspect of this embodiment, the assay amount of the test sample is rate limiting.

Aspects of the present disclosure can also be described as follows:
1. A composition comprising a carrier linked to a mobile linker linked to a SNAP-25 antigen having a carboxyl terminus at the P ₁ residue of the BoNT / A cleavage site cleavable bond.

2. BoNT / A cleavage site scissile bond _{P 1} residue 1 of the composition is glutamine or lysine.

  3. The composition of 1, wherein the SNAP-25 antigen comprises SEQ ID NO: 147.

  4. The composition of 1, wherein the mobile linker and SNAP-25 antigen amino acid sequence is SEQ ID NO: 38 or SEQ ID NO: 46.

5. SNAP-25 cleavage BoNT / A cleavage site scissile isolated α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at _{the P 1} residue from binding from the product.

6. α-SNAP-25 antibody has an association rate constant of less than 1 × 10 ¹ M ⁻¹ s ^{−1 for an} epitope that does not contain the carboxyl terminal glutamine of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. And 5 isolated α-SNAP-25 antibodies, wherein the α-SNAP-25 antibody has an equilibrium dissociation constant of less than 0.450 nM with respect to the epitope.

  7. The isolated α-SNAP-25 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, and SEQ ID NO: 82. A heavy chain variable region; and 5 having a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90 and SEQ ID NO: 92. Isolated α-SNAP-25 antibody.

8. The isolated α-SNAP-25 antibody at least SEQ ID NO: 93 of the _V H CDRl, SEQ ID NO: 94 of the _V H CDRl, SEQ ID NO: 95 _V H CDRl, SEQ ID NO: 118 of the _V H CDRl, 5. 5 isolated α-SNAP-25 antibodies comprising the V _H CDR1 of SEQ ID NO: 119 or the V _H CDR1 of SEQ ID NO: 120.

9. The isolated α-SNAP-25 antibody at least SEQ ID NO: 96 of the _V H CDR2, SEQ ID NO: 97 _V H CDR2, SEQ ID NO: 98 _V H CDR2, SEQ ID NO: 99 _V H CDR2, 5. 5 isolated α-SNAP-25 antibodies comprising V _H CDR2 of SEQ ID NO: 121, V _H CDR2 of SEQ ID NO: 122 or V _H CDR2 of SEQ ID NO: 123.

10. The isolated α-SNAP-25 antibody at least SEQ ID NO: 100 of the _V H CDR3, SEQ ID NO: 101 of the _V H CDR3, SEQ ID NO: 102 _V H _V H CDR3 or SEQ ID NO: 124 _{V H} 5 isolated α-SNAP-25 antibodies comprising CDR3.

11. The isolated α-SNAP-25 antibody at least SEQ ID NO: 103 _V L CDRl, SEQ ID NO: 104 _V L CDRl, SEQ ID NO: 105 _V L CDRl, SEQ ID NO: 106 _V L CDRl, SEQ ID NO: 107 V _L CDR1, SEQ ID NO: 125 V _L CDR1, SEQ ID NO: 126 V _L CDR1, SEQ ID NO: 127 V _L CDR1, SEQ ID NO: 128 V _L CDR1 or SEQ ID NO: 129 5 isolated α-SNAP-25 antibodies comprising V _L CDR1.

12. The isolated α-SNAP-25 antibody at least SEQ ID NO: 108 _V L CDR2, SEQ ID NO: 109 _V L CDR2, SEQ ID NO: 110 _V L CDR2, SEQ ID NO: 111 _V L CDR2, or 5. Isolated α-SNAP-25 antibody comprising the V _L CDR2 of SEQ ID NO: 112.

13. The isolated α-SNAP-25 antibody at least SEQ ID NO: 113 _V L CDR3, SEQ ID NO: 114 _V L CDR3, SEQ ID NO: 115 _V L CDR3, SEQ ID NO: 116 of the _V L CDR3 or 5. 5 isolated α-SNAP-25 antibodies comprising V _L CDR3 of SEQ ID NO: 117.

  14. A heavy chain variable region wherein the isolated α-SNAP-25 antibody comprises SEQ ID NO: 93, SEQ ID NO: 121 and SEQ ID NO: 100; and SEQ ID NO: 105, SEQ ID NO: 110 and SEQ ID NO: 5. 5 isolated α-SNAP-25 antibodies comprising a light chain variable region comprising 115.

  15. The isolated α-SNAP-25 antibody is SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 147 or SEQ ID NO: 5 isolated α-SNAP-25 antibodies that selectively bind to 148 SNAP-25 epitopes.

  16. The isolated α-SNAP-25 antibody is selective for the SNAP-25 epitope of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. 5 isolated α-SNAP-25 antibodies that bind to.

17. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line are highly susceptible to BoNT / A poisoning by BoNT / A ; (b) treated with SNAP-25 comprising the cleavage product SNAP-25 component having a carboxyl-terminus at _{the P 1} residue from cells BoNT / a cleavage site scissile bond can be isolated; (c ) Contacting the SNAP-25 component with the α-SNAP-25 antibody, where the α-SNAP-25 antibody is carboxyl-terminal at the P1 residue of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. And (d) detecting the presence of an antibody-antigen complex comprising an α-SNAP-25 antibody and a SNAP-25 cleavage product. When; wherein the antibody - detection by antigen complex is indicative of BoNT / A activity; a method of detecting BoNT / A activity comprising the steps.

18. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line are sensitive to BoNT / A poisoning by BoNT / A ; (b) treated with SNAP-25 comprising the cleavage product SNAP-25 component having a carboxyl-terminus at _{the P 1} residue from cells BoNT / a cleavage site scissile bond can be isolated; (c ) Contacting the SNAP-25 component with an α-SNAP-25 antibody linked to a solid support, where the α-SNAP-25 antibody is capable of cleaving the BoNT / A cleavage site from the SNAP-25 cleavage product. Binds to an epitope comprising a carboxyl terminus at the P1 residue of binding; and (d) an antibody-antigen comprising an α-SNAP-25 antibody and a SNAP-25 cleavage product Detecting the presence of a coalescing; wherein the antibody - detection by antigen complex is indicative of BoNT / A activity; a method of detecting BoNT / A activity comprising the steps.

19. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line are highly susceptible to BoNT / A poisoning by BoNT / A ; (b) treated with SNAP-25 comprising the cleavage product SNAP-25 component having a carboxyl-terminus at _{the P 1} residue from cells BoNT / a cleavage site scissile bond can be isolated; (c ) Immobilizing the SNAP-25 component to the solid support; (d) contacting the SNAP-25 component with the α-SNAP-25 antibody, wherein the α-SNAP-25 antibody generates a SNAP-25 cleavage. Binds to an epitope comprising the carboxyl terminus at the P1 residue of the BoNT / A cleavage site cleavable linkage from the product; and (e) α-SNAP-25 antibody and SNAP-2 Detecting BoNT / A activity comprising detecting the presence of an antibody-antigen complex comprising a cleavage product; wherein detection by the antibody-antigen complex is an indicator of BoNT / A activity; how to.

20. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line can take up BoNT / A; (b) isolating the SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus from cells treated with _{the P 1} residue of the BoNT / a cleavage site scissile bond; (c) SNAP-25 Contacting the component with an α-SNAP-25 antibody, wherein the α-SNAP-25 antibody comprises a carboxyl terminus at the P1 residue of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. Binding to the epitope; and (d) detecting the presence of an antibody-antigen complex comprising the α-SNAP-25 antibody and the SNAP-25 cleavage product; - detection by antigen complex is indicative of BoNT / A activity; a method of detecting BoNT / A activity comprising the steps.

21. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line can take up BoNT / A; (b) isolating the SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus from cells treated with _{the P 1} residue of the BoNT / a cleavage site scissile bond; (c) SNAP-25 Contacting the component with an α-SNAP-25 antibody linked to a solid support, wherein the α-SNAP-25 antibody is a P1 residue of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. Binding to an epitope comprising a carboxyl terminus with a group; and (d) the presence of an antibody-antigen complex comprising an α-SNAP-25 antibody and a SNAP-25 cleavage product. To output; wherein the antibody - detection by antigen complex is indicative of BoNT / A activity; a method of detecting BoNT / A activity comprising the steps.

22. (a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line can take up BoNT / A; (b) isolating the SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus from cells treated with _{the P 1} residue of the BoNT / a cleavage site scissile bond; (c) SNAP-25 Immobilizing the component to a solid support; (d) contacting the SNAP-25 component with the α-SNAP-25 antibody, wherein the α-SNAP-25 antibody is BoNT from the SNAP-25 cleavage product. Binds to an epitope comprising the carboxyl terminus at the P1 residue of the / A cleavage site cleavable linkage; and (e) α-SNAP-25 antibody and SNAP-25 cleavage product Detecting the presence of the antigen complex - an antibody which Nde; wherein the antibody - detection by antigen complex is indicative of BoNT / A activity; a method of detecting BoNT / A activity comprising the steps.

23. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibodies; (b) cells from established cell lines; treatment with test sample, wherein the established cells from a cell line is highly sensitive to BoNT / a intoxication; (c) processing carboxyl at the P ₁ residue of the BoNT / a cleavage site scissile bond from a cell Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) contacting the SNAP-25 component with an α-SNAP-25 antibody, wherein α-SNAP- 25 antibody binds an epitope comprising a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product; (e) α-SNAP- 25 antibody And detecting the presence of an antibody-antigen complex comprising a SNAP-25 cleavage product; (f) repeating steps be with a negative control sample instead of the test sample, wherein the negative control sample is BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; and (g) the amount of antibody-antigen complex detected in step e detected in step f. The amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is A method for determining BoNT / A immunoresistance in a mammal comprising the step of: -indicating the presence of a BoNT / A neutralizing antibody.

24. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibodies; (b) cells from established cell lines; treatment with test sample, wherein the established cells from a cell line is highly sensitive to BoNT / a intoxication; (c) processing carboxyl at the P ₁ residue of the BoNT / a cleavage site scissile bond from a cell Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) contacting the SNAP-25 component with an α-SNAP-25 antibody linked to a solid support. it, where alpha-SNAP-25 antibody binds an epitope comprising a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product; (e) detecting the presence of an antibody-antigen complex comprising an α-SNAP-25 antibody and a SNAP-25 cleavage product; (f) repeating steps be with a negative control sample instead of the test sample. The negative control sample comprises BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; and (g) the amount of antibody-antigen complex detected in step e To the amount of antibody-antigen complex detected in step f, wherein the antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f. A less detectable amount is an indicator of the presence of α-BoNT / A neutralizing antibody; a method of determining BoNT / A immunoresistance in a mammal comprising the step of:

25. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibodies; (b) cells from established cell lines; treatment with test sample, wherein the established cells from a cell line is highly sensitive to BoNT / a intoxication; (c) processing carboxyl at the P ₁ residue of the BoNT / a cleavage site scissile bond from a cell Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) immobilizing the SNAP-25 component to a solid support; (e) a SNAP-25 component contacting the alpha-SNAP-25 antibody, wherein the alpha-SNAP-25 antibody comprising a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product (F) detecting the presence of an antibody-antigen complex comprising α-SNAP-25 antibody and SNAP-25 cleavage product; (g) a negative control sample instead of the test sample In step b-f, wherein the negative control sample comprises BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; and (h) in step f Comparing the amount of antibody-antigen complex detected with the amount of antibody-antigen complex detected in step (g), here relative to the amount of antibody-antigen complex detected in step g. A lower detectable amount of the antibody-antigen complex detected in step f is an indicator of the presence of α-BoNT / A neutralizing antibody; determining BoNT / A immunoresistance in a mammal comprising the step Method.

26. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibody; (b) cells from an established cell line treatment with test sample, wherein the cells from cell lines established in can take in a BoNT / a; (c) a carboxyl from cells treated with the P ₁ residue of the BoNT / a cleavage site scissile bond Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) contacting the SNAP-25 component with an α-SNAP-25 antibody, wherein α-SNAP- 25 antibody binds an epitope comprising a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product; (e) α-SNAP- 25 Detecting the presence of an antibody-antigen complex comprising the SNAP-25 cleavage product and (f) repeating step be with a negative control sample instead of the test sample, the negative control sample Comprises BoNT / A and serum known to be free of α-BoNT / A neutralizing antibodies; and (g) detecting the amount of antibody-antigen complex detected in step e in step f. The detected amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f, A method of determining BoNT / A immunoresistance in a mammal comprising the step of: a step comprising the step of: α-BoNT / A neutralizing antibody.

27. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibody; (b) cells from an established cell line treatment with test sample, wherein the cells from cell lines established in can take in a BoNT / a; (c) a carboxyl from cells treated with the P ₁ residue of the BoNT / a cleavage site scissile bond Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) contacting the SNAP-25 component with an α-SNAP-25 antibody linked to a solid support. it, where alpha-SNAP-25 antibody binds an epitope comprising a carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product; ( e) detecting the presence of an antibody-antigen complex comprising α-SNAP-25 antibody and SNAP-25 cleavage product; (f) repeating step be with a negative control sample instead of the test sample. The negative control sample comprises BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; and (g) the antibody-antigen complex detected in step e Is compared to the amount of antibody-antigen complex detected in step f, wherein the antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f Detecting a lower amount of the body is an indication of the presence of α-BoNT / A neutralizing antibody; a method for determining BoNT / A immunoresistance in a mammal comprising the step of:

28. (a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibody; (b) cells from an established cell line treatment with test sample, wherein the cells from cell lines established in can take in a BoNT / a; (c) a carboxyl from cells treated with the P ₁ residue of the BoNT / a cleavage site scissile bond Isolating a SNAP-25 component comprising a terminal SNAP-25 cleavage product; (d) immobilizing the SNAP-25 component to a solid support; (e) a SNAP-25 component contacting the alpha-SNAP-25 antibody, here alpha-SNAP-25 antibody carboxyl-terminus at the _{P 1} residue of the BoNT / a cleavage site scissile bond from SNAP-25 cleavage product Binding to the epitope comprising; (f) detecting the presence of an α-SNAP-25 antibody and an antibody-antigen complex comprising the SNAP-25 cleavage product; (g) a negative control instead of the test sample Repeating step b-f with the sample, the negative control sample comprising BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; and (h) step f Comparing the amount of antibody-antigen complex detected in step with the amount of antibody-antigen complex detected in step g, wherein the step is relative to the amount of antibody-antigen complex detected in step g. A lower detectable amount of antibody-antigen complex detected in f is an indicator of the presence of α-BoNT / A neutralizing antibody; a method for determining BoNT / A immunoresistance in a mammal comprising the step of: .

  29. The method of 17-22 and 23-25, wherein the cells are sensitive to BoNT / A poisoning by BoNT / A by about 500 pM or less, by about 400 pM or less, by about 300 pM or less, by about 200 pM or less, by about 100 pM or less.

  30. The method of 20-22 and 26-28, wherein the cells can take up BoNT / A up to about 500 pM, up to about 400 pM, up to about 300 pM, up to about 200 pM, up to about 100 pM.

  31. The method of 17-22, wherein the sample comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, 100 fg or less, 10 fg or less, or 1 fg or less of BoNT / A.

  32. A sample comprising BoNT / A comprising about 100 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM or less, about 1 pM or less, about 100 fM or less, about 10 fM or less, or about 1 fM or less 17-22 the method of.

  33. The method of 17-28, wherein the α-SNAP-25 antibody is 5-16 isolated α-SNAP-25 antibody.

  34. The method of 17-28, wherein the presence of the antibody-antigen complex is detected by immunoblot analysis, immunoprecipitation analysis, ELISA or sandwich ELISA.

  35. Immune-based methods are at least 3: 1, at least 5: 1, at least 10: 1, at least 20: 1, at least 50: 1 or at least 100: 1 signal to noise with respect to the lower asymptote 17-28 method having a ratio.

  36. The immune-based method is at least 10: 1, at least 20: 1, at least 50: 1, at least 100: 1, at least 200: 1, at least 300: 1, at least 400: with respect to the higher asymptote 1. The method of 17-28 having a signal to noise ratio of at least 500: 1 or at least 600: 1.

37. Immune-based methods detect EC ₅₀ activity of for example at least 100 ng, at least 50 ng, at least 10 ng, at least 5 ng, at least 100 pg, at least 50 pg, at least 10 pg, at least 5 pg, at least 100 fg, at least ₅₀ fg, at least 10 fg or at least 5 fg 17-28 methods that can.

38. Immune-based methods can detect, for example, EC ₅₀ activity of at least 10 nM, at least 5 nM, at least 100 pM, at least 50 pM, at least 10 pM, at least 5 pM, at least 100 fM, at least 50 fM, at least 10 fM, at least 5 fM or at least 1 fM. The method of 17-28.

  39. The method of 17-28, wherein the immune-based method has a BoNT / A LOD of 10 pg or less, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less .

  40. The method according to 17-28, wherein the immune-based method has a BoNT / A LOD of, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less. .

  41. The method of 17-28, wherein the immune-based method has a BoNT / A LOQ of 10 pg or less, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, 1 pg or less .

  42. The method of 17-28, wherein the immune-based method has a BoNT / A LOQ of, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less. .

  43. BoNT / A for which the immune-based method is sufficiently active is reduced to 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less of the activity of fully active BoNT / A or 17-28 methods that can be distinguished from partially active BoNT / A having 10% or less.

Screening Candidate Cell Lines The following examples are established cells that are sensitive to BoNT / A intoxication or have the BoNT / A uptake capacity required for the methods of detecting BoNT / A activity disclosed herein. Illustrates how to identify strains.
1. Growth of candidate cell line stock cultures In order to grow cell lines, 162 cm ² tissue culture containing 30 mL of appropriate growth medium (see Table 1) with an appropriate density of cells from the cell line under test. The flask was seeded and grown under 5% or 10% carbon dioxide in a 37 ° C. incubator until the cells reached the desired density.

2. Single Dose Screening of Candidate Cell Lines Using 1 nM BoNT / A One parameter that is tested to improve the sensitivity of cell-based assays is good for taking up clostridial neurotoxin and adhering to the substrate surface It was to identify an appropriate cell line that exhibited receptivity. Initially the cell line was tested for its ability to take up 1 nM BoNT / A and adhere to the surface. To determine if the cell line can take up 1 nM BoNT / A, contain the appropriate density of cells from the stock culture of the cell line under test, and 1 mL of the appropriate serum growth medium (Table 1). Seeds were seeded in wells of 24-well tissue culture plates. Cells were grown under 5% carbon dioxide in a 37 ° C. incubator until the cells reached the desired density (approximately 18-24 hours). Growth medium is aspirated from each well and replaced with either 1) fresh growth medium containing no toxin (untreated cell line) or 2) fresh growth medium containing 1 nM BoNT / A complex (treated cell line). It was. After overnight incubation, the cells were washed by aspirating the growth medium and rinsing each well with 200 μL of 1 × PBS. To harvest the cells, the cells were lysed by aspirating 1 × PBS, adding 50 μL of 2 × SDS loading buffer, transferring the lysate to a clean tube, and heating the sample to 95 ° C. for 5 minutes.

  An aliquot from each collected sample was analyzed by Western blot to detect for the presence of both SNAP-25 uncleaved substrate and SNAP-25 cleavage product. In this analysis, a 12 μL aliquot of the collected sample was subjected to MOPS polyacrylamide gel electrophoresis using NuPAGE® Novex 12% Bis-Tris precast polyacrylamide gel (Invitrogen Inc., Carlsbad, Calif.) Under denaturing reducing conditions. Separation by electrophoresis. Separated peptides were converted from gel to polyvinylidene fluoride (PVDF) membrane (Invitrogen Inc.) by Western blotting using a Trans-Blot® SD semi-dry electrophoresis transfer cell apparatus (Bio-Rad Laboratories, Hercules, Calif.). , Carlsbad, CA). Tris buffered saline (TBS) (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), By incubating at room temperature for 2 hours in a solution containing 1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate), 2% bovine serum albumin (BSA), 5% nonfat dry milk The PVDF membrane was blocked. Blocked membrane 1) 1: 5,000 dilution of α-SNAP-25 mouse monoclonal antibody (SMI-81; Sternberger Monoclonals Inc., Lutherville, MD) as primary antibody; or 2) S9684 α-SNAP as primary antibody TBS containing either 1: 5,000 dilution of -25 rabbit polyclonal antiserum (Sigma, St. Louis, MO), 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan mono Laureate) and incubated overnight at 4 ° C. in 2% BSA and 5% nonfat dry milk. Both α-SNAP-25 mouse monoclonal and rabbit polyclonal antibodies can detect both SNAP-25 uncleaved substrate and SNAP-25 cleavage products, and as parameters to assess the amount of BoNT / A incorporation, It becomes possible to evaluate the overall SNAP-25 expression in each cell line and the percentage of SNAP-25 cleaved after BoNT / A treatment. Blots probed with primary antibody were washed 3 times with TBS, TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) for 15 minutes each time. Washed membranes 1) 1: 10,000 of goat polyclonal anti-mouse immunoglobulin G, heavy and light chain (IgG, H + L) antibodies (Zymed, South San Francisco, Calif.) Conjugated to horseradish peroxidase as a secondary antibody Dilution; or 2) 1: 10,000 dilution of goat polyclonal anti-rabbit immunoglobulin G, heavy and light chain (IgG, H + L) antibody (Zymed, South San Francisco, Calif.) Conjugated to horseradish peroxidase as a secondary antibody Incubated in TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate), 2% BSA and 5% nonfat dry milk for 2 hours at room temperature. Blots probed with secondary antibody were washed 3 times in TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) for 15 minutes each time. Signal detection of labeled SNAP-25 product was visualized using the ECL Plus ™ Western blot detection system (GE Healthcare, Amersham Biosciences, Piscataway, NJ), and the membrane was imaged and the percent cleaved was measured by Typhoon 9410 Quantification was performed with a variable format imaging device and imaging device analysis software (GE Healthcare, Amersham Biosciences, Piscataway, NJ). The choice of pixel size (100 to 200 pixels) and PMT voltage settings (350 to 600, usually 400) depended on the individual blot. Table 2 shows the cell lines in which SNAP-25 cleavage products were detected when treated with 1 nM BoNT / A. The following cell lines exhibited both 1 nM BoNT / A uptake and proper attachment to the substrate surface: BE (2) -M17, IMR-32, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SY5Y, SiMa and SK-N-BE (2) -C.

To determine if the cell line can adhere to the surface, the appropriate density of cells from the stock culture of the cell line under test is transferred to a 24-well tissue culture plate containing 1 mL of the appropriate growth medium (Table 1). I went to the well. Cells were grown under 5% carbon dioxide in a 37 ° C. incubator until the cells reached the desired density (approximately 18-24 hours). Cell attachment was assessed by the percentage of cells attached to the bottom well surface of the tissue plate, relative to the total number of cells seeded. Cell lines CHP-126, IMR-32, LA-N-1, MC-IXC, NG115-401L, SK-N-BE (2) -C, SK-N-F1 and SK-N-MC are less than 50% (Table 2), each cell line was considered unsuitable. All other cell lines tested exhibited appropriate adhesion characteristics (Table 2).

Evaluation of Growth Conditions for Neurotoxin Uptake in Candidate Cell Lines The following examples are for established cell lines that maximize sensitivity to BoNT / A intoxication or have BoNT / A uptake capacity. Illustrates how to determine growth conditions for
1. Effect of cell differentiation on neurotoxin uptake of candidate cell lines To determine whether cell differentiation improved neurotoxin uptake, cell lines exhibiting 1 nM BoNT / A uptake were transferred to serum-free medium. Differentiation was induced. Appropriate density of cells from the stock culture of the cell line under test is transferred to 2 mM GlutaMAX ™ I, 0.1 mM non-essential amino acid, 10 mM HEPES, 1 mM sodium pyruvate, 100 units / mL penicillin and 100 μg with Earl salt. The wells were plated in 24-well tissue culture plates containing 1 mL of serum-free medium containing minimal essential medium with / mL streptomycin. These cells are differentiated under approximately 5% carbon dioxide in a 37 ° C. incubator until the cells are differentiated as assessed by standard and routine morphological criteria such as growth inhibition and neurite outgrowth (approximately 2 to 3 Day). As a control, cells of the appropriate density were seeded from a stock culture of the cell line under test into wells of a 24-well tissue culture plate containing 1 mL of the appropriate growth medium (Table 1). These undifferentiated control cells were grown under 5% carbon dioxide in a 37 ° C. incubator until the cells reached the desired density (approximately 18-24 hours). Medium from both differentiated and undifferentiated control cultures is aspirated from each well and fresh containing either 0 (untreated sample), 0.1 nM, 0.3 nM or 1 nM BoNT / A complex. The medium was replaced. After overnight incubation, the cells were washed and harvested as described in Example 1.

Example 1 except that collected samples are separated by SDS-PAGE using a 12% 26 well Criterion gel (Bio-Rad Laboratories, Hercules, CA) to detect the presence of SNAP-25 cleavage products. An aliquot from each collected sample was analyzed by Western blot as described in and rabbit polyclonal α-SNAP-25 ₁₉₇ antibody serum was used as the primary antibody (see Example 4). Table 3 shows the cell lines that exhibited the SNAP-25 cleavage product when treated with 0.1 nM BoNT / A. Of the cell lines tested, only SiMa and Neuro-2a cell lines exhibited uptake of 0.1 nM BoNT / A in an undifferentiated state. However, in addition to SiMa and Neuro-2a, cell lines N18, LA1-55n, PC12 and SH-SY5Y all showed uptake of 0.1 nM BoNT / A.

2. Effect of ganglioside treatment on neurotoxin uptake of differentiated candidate cell lines exhibits uptake of 1 nM BoNT / A to determine if treatment that improves low affinity binding of neurotoxin improves neurotoxin uptake Differentiated cell lines were treated with ganglioside GT1b. Appropriate density of cells from the stock culture of the cell line under test is 24 wells containing serum-free medium as described above with or without 25 μg / mL GT1b (Alexis Biochemicals, San Diego, Calif.). Seeded into wells of tissue culture plate. These cells were incubated in 5% carbon dioxide in a 37 ° C. incubator until the cells differentiated as assessed by standard and routine morphological criteria as described above. Medium is aspirated from each well and 0 (untreated sample), 1.9 pM, 3.7 pM, 7.4 pM, 14.8 pM, 29.7 pM, 59.4 pM, 118.8 pM, 237.5 pM, 574 pM, Replaced with fresh serum-free medium containing either 950 pM and 1900 pM BoNT / A complex. Cell lines were incubated at two different times, 24 hours and 48 hours. Following toxin incubation, cells were washed and harvested as described in Example 1.

Example 1 except that collected samples are separated by SDS-PAGE using a 12% 26 well Criterion gel (Bio-Rad Laboratories, Hercules, CA) to detect the presence of SNAP-25 cleavage products. An aliquot from each collected sample was analyzed by Western blot as described in and rabbit polyclonal α-SNAP-25 ₁₉₇ antibody serum was used as the primary antibody (see Example 4). Table 4 shows the effect of ganglioside treatment on the ability of differentiated cell lines to take up BoNT / A. These results indicate the lowest concentration of BoNT / A that produces a detectable band of SNAP-25 cleavage product in Western blots.

3. Development of serum-free media with cell differentiation properties that enhance neurotoxin uptake of candidate cell lines To determine whether treatment improvements that induce cell differentiation can improve neurotoxin uptake, SiMa, Neuro-2a And PC12 cell lines were grown in various serum-free media to induce differentiation. Cells of the appropriate density from the stock culture of the cell line under test were seeded into wells of 24-well tissue culture plates containing 1 mL of various test serum-free media. Parameters tested were: 1) Effect of different basal media on BoNT / A uptake (MEM and RPMI1649); 2) Effect of presence or absence of neurotrophic factor on BoNT / A uptake (N2 and B27 supplements); 3 ) Effects of presence or absence of differentiation factors on BoNT / A uptake (retinoic acid and nerve growth factor); and 4) effects of presence or absence of serum on BoNT / A uptake (serum-free medium and reduced serum usage). Medium). As a control, cells of the appropriate density from the stock culture of the cell line under test were transferred to a control serum-free medium (minimum essential medium, 2 mM GlutaMAX ™ I with Earl's salt, 0.1 mM non-essential amino acid, 10 mM HEPES, We seeded wells of 24-well tissue culture plates containing 1 mL of 1 mM sodium pyruvate, 100 units / mL penicillin and 100 μg / mL streptomycin). These cells are differentiated under approximately 5% carbon dioxide in a 37 ° C. incubator until the cells are differentiated as assessed by standard and routine morphological criteria such as growth inhibition and neurite outgrowth (approximately 2 to 3 Day). Medium is aspirated from each well and 0 (untreated sample), 0.005 pM, 0.015 pM, 0.05 pM, 0.14 pM, 0.42 pM, 1.2 pM, 3.7 pM, 11 pM, 33 pM, 100 pM and Replaced with fresh serum-free medium containing either 300 pM BoNT / A complex. In addition, differentiated cells were treated with BoNT / A for 24 hours followed by medium change and incubation for 48 hours without toxin in fresh medium to allow accumulation of SNAP-25 cleavage products. Cells were then washed and harvested as described in Example 1.

  Example 1 except that collected samples are separated by SDS-PAGE using a 12% 26 well Criterion gel (Bio-Rad Laboratories, Hercules, CA) to detect the presence of SNAP-25 cleavage products. An aliquot from each collected sample was analyzed by Western blot and α-SNAP-25 rabbit polyclonal serum was used (see Example 4). The most optimized media determined for each cell line is shown in Table 5. Table 6 shows the lowest amount of the lowest amount of SNAP-25 cleavage product detected when the cell line was grown in this optimized serum-free medium. The use of optimized serum-free medium resulted in the detection of a BoNT / A activity signal with an acceptable signal-to-noise ratio in LA1-55n, Neuro-2a, PC-12 and SiMa cell lines (FIG. 2). For example, optimized differentiation conditions resulted in a 5-fold increase for Neuro-2a and PC12 cells and a nearly 50-fold increase for SiMa cells compared to control serum-free medium in SNAP-25 cleavage product detection. In addition, a minimum signal to noise ratio of 3: 1 for the lower asymptote and 10: 1 for the upper asymptote is required to develop a robust assay that can be validated. When comparing the signal detected from the 1.2 pM dose with the background signal of 0 pM BoNT / A, all optimized cell lines, except LA-1-55n, gave at least 3 for the lower asymptote. A signal-to-noise ratio of 1 was provided (Figure 2). In addition, when the signal from the 300 pM dose was compared to the background signal of 0 pM BoNT / A, all optimized cell lines provided a signal to noise ratio of at least 100: 1 with respect to the upper asymptote ( Figure 2). These results indicate that since the assay detected the presence of pM amounts of BoNT / A, any of these cell lines could be used to detect BoNT / A activity as disclosed herein. It is shown that it can develop immune-based methods of

BoNT / A to the SNAP-25 epitope having a free carboxyl-terminus at the _{P 1} residue of the cleavage site scissile bond following examples the development of alpha-SNAP-25 monoclonal antibodies that selectively bind the BoNT / A cleavage site cleavable Illustrates how to make an α-SNAP-25 monoclonal antibody capable of selectively binding to a SNAP-25 epitope having a carboxyl terminus at the binding P ₁ residue.
1. alpha-SNAP-25 monoclonal alpha-SNAP-25 that binds an epitope comprising a carboxyl-terminus at _{P 1} residue from the BoNT / A cleavage site scissile bond from creating SNAP-25 cleavage product monoclonal antibodies To develop the antibody, the 13-residue peptide CDSNKTRIDEEANQ _COOH (SEQ ID NO: 38) was designed as a SNAP-25 cleavage product antigen. This peptide is an N-terminal cysteine residue for conjugation to amino acids 186-197 of human SNAP-25 (SEQ ID NO: 5) with a mobile linker region and KLH and a carboxylated C-terminal glutamine (SEQ ID NO: 38) Comprising. Generation of monoclonal antibodies against selected and unique peptide sequences controls epitopes that allow the identification of specific subpopulations of proteins within a pool of closely related isoforms. A Blast search showed that this peptide has high homology only to SNAP-25 and has little potential for cross-reactivity with other proteins in neuronal cells. The sequence was also carefully scrutinized using computer algorithms to determine the hydrophobic hydrophilicity index, protein surface probability, mobility region and preferred secondary structure, followed by the appropriate orientation and presentation of the selected peptide sequence. . Peptides were synthesized and conjugated to keyhole limpet hemocyanin (KLH) to increase immunogenicity. Six Balb / c mice were immunized with this peptide, and after 3 immunizations in about 8 weeks, the mice were bled for testing. The blood was allowed to clot by incubating at 4 ° C. for 60 minutes. The coagulated blood was centrifuged at 10,000 × g for 10 minutes at 4 ° C. to pellet cell debris. The resulting serum was aliquoted into 50 μL aliquots and stored at −20 ° C. until needed.

With plans similar based on other SNAP-25 antigens disclosed herein, include a carboxyl-terminus at the _{P 1} residue from the BoNT / A cleavage site scissile bond from SNAP-25 cleavage product An α-SNAP-25 monoclonal antibody that binds to the epitope consisting of For example, the SNAP-25 antigen of SEQ ID NO: 45 can be conjugated to KLH instead of the SNAP-25 antigen of SEQ ID NO: 38. As another illustration, amino acids 186-197 of human SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 are SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44.

2. α-SNAP-25 SNAP- 25 antigen selectively bind alpha-SNAP-25 monoclonal capable of having a carboxyl-terminus at _{the P 1} residue of the screening BoNT / A cleavage site scissile bond for the presence of monoclonal antibodies To determine the presence of the antibody, a comparative ELISA and cell-based cleavage assay was performed using the extracted mouse serum. Two fusion proteins were constructed for comparative ELISA: BirA-HisTag®-SNAP-25 _134-197 of SEQ ID NO: 48 and BirA-HisTag®-SNAP-25 of SEQ ID NO: 49. _134-206 . BirA-HisTag®-SNAP-25 _134-197 is a natural biotinylated 16 amino acid of SEQ ID NO: 50 linked at the amino terminus to a SNAP-25 peptide comprising amino acids 134-197 of SEQ ID NO: 5 BirA peptide was included. BirA-HisTag®-SNAP-25 _134-206 is a natural biotinylated 16 amino acid BirA of SEQ ID NO: 50 linked at the amino terminus to a SNAP-25 peptide comprising amino acids _134-206 of SEQ ID NO: 5. Peptide. These two substrates were suspended in 1 × PBS at concentrations of 10 μg / mL BirA-HisTag®-SNAP-25 _134-197 and BirA-HisTag®-SNAP-25 _134-206 . BirA-HisTag®-SNAP-25 _134-197 and BirA-HisTag®-SNAP-25 _134-206 are coated on separate plates by adding approximately 100 μL of the appropriate substrate solution, and Plates were incubated for 1 hour at room temperature. Washed plates were used from 1:10 to 1: 100 of antibody-containing serum derived from one of six immunized mice (Mouse 1, Mouse 2, Mouse 3, Mouse 4, Mouse 5 and Mouse 6). Incubated for 1 hour at 37 ° C. in 0.5% BSA in 1 × TBS containing dilution. Plates probed with primary antibody were washed 4 times for 5 minutes each in 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Washed plates were washed at 37 ° C. in 1 × TBS containing a 1: 10,000 dilution of goat polyclonal anti-mouse IgG antibody (Pierce Biotechnology, Rockford, IL) conjugated to horseradish peroxidase as a secondary antibody. Incubated for hours. Plates probed with secondary antibody were washed 4 times in 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Color detection of labeled SNAP-25 product was visualized by color detection using an ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, IL). BirA-HisTag®-SNAP-25 _134-197 coated plates developed a yellow color, while BirA-HisTag®-SNAP-25 _134-206 coated plates did not develop color, thereby It was shown that α-SNAP-25 antibody preferentially recognizes the SNAP-25 ₁₉₇ cleavage product. The results show that 3 out of 6 mice used for immunization (mouse 2, mouse 3 and mouse 4) were carboxyl-terminated with a higher titer and a P ₁ residue of the BoNT / A cleavage site cleavable linkage. Had additional specificity for the SNAP-25 antigen.

These results were confirmed using an ELISA light chain activity assay. A 96-well Reacti-Bind streptavidin-coated plate (Pierce Biotechnology, Rockford, IL) was prepared by adding approximately 100 μL of the following substrate solution: rows AC with 12 different concentrations of BirA-HisTag®. ) -SNAP-25 _134-197 coated with 100 [ _{mu] L} ; row D-H was coated with 10 [mu] g / mL BirA-HisTag <(R)>-SNAP-25 _134-206 100 [ _{mu] L.} The plate was washed by aspirating the substrate solution and rinsing each well 3 times with 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). A dilution of BoNT / A was prereduced for 20 minutes at 37 ° C. in BoNT / A incubation buffer (50 mM HEPES, pH 7.4, 1% fetal calf serum, 10 μM ZnCl ₂ , 10 mM dithiothreitol) and prereduced BoNT / A 100 μL was added to the substrate coated plate and incubated at 37 ° C. for 90 minutes. BoNT / A incubation buffer was aspirated and BoNT / A was rinsed 3 times with 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). The treatment plate was washed. Washed plates were incubated for 1 hour at 37 ° C. in 0.5% BSA in 1 × TBS containing a 1:10 to 1: 100 dilution of antibody-containing serum under test. Plates probed with primary antibody were washed 4 times for 5 minutes each in 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Washed plates were washed at 37 ° C. in 1 × TBS containing a 1: 10,000 dilution of goat polyclonal anti-mouse IgG antibody (Pierce Biotechnology, Rockford, IL) conjugated to horseradish peroxidase as a secondary antibody. Incubated for hours. Plates probed with secondary antibody were washed 4 times in 200 μL of TBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Color detection of labeled SNAP-25 product was visualized by color detection using an ImmunoPure TMB substrate kit (Pierce Biotechnology, Rockford, IL). Antibody correlated sera derived from all 6 immunized mice (mouse 1, mouse 2, mouse 3, mouse 4, mouse 5 and mouse 6) were used to correlate with the presence of SNAP-25 ₁₉₇ cleavage product Color development was detected in the BoNT / A-treated sample but not in the untreated control. Thus, by comparative ELISA analysis, 3 of the mice used for immunization (mouse 2, mouse 3 and mouse 4) were carboxyl-terminated with a higher titer and a P ₁ residue of the BoNT / A cleavage site cleavable linkage. Had additional specificity for the SNAP-25 antigen.

For cell-based cleavage assays, the appropriate density of PC12 cells was plated on 60 mm ² tissue culture plates containing 3 mL of appropriate serum medium (Table 1). Cells were grown under 5% carbon dioxide in a 37 ° C. incubator until the cells reached the appropriate density. 250 μL of OPTI-MEM reduced serum medium containing 15 μL of LipofectAmine 2000 (Invitrogen Inc., Carlsbad, Calif.) Incubated for 5 minutes at room temperature was added to the pQBI-25 / GFP-BoNT / A-LC expression construct (SEQ ID NO: 51). A 500 μL transfection solution was prepared by adding to 250 μL OPTI-MEM reduced serum medium containing 10 μg. The pQBI-25 / GFP-BoNT / A-LC expression construct comprises a pQBI-25 expression vector (Qbiogene Inc., Carlsbad, Calif.), whose promoter element contains the GFP-BoNT / A light chain of SEQ ID NO: 52. It is operably linked to the encoding polynucleotide. The transfection mixture was incubated for approximately 20 minutes at room temperature. The medium was replaced with fresh unsupplemented medium and 500 μL of transfection solution was added to the cells. Cells were then incubated for approximately 6-18 hours under 5% carbon dioxide in a 37 ° C. incubator. Cells were washed and harvested as described in Example 2. To detect the presence of the SNAP-25 ₁₉₇ cleavage product, the primary antibody used was a 1: 1,000 dilution of antibody-containing serum and the secondary antibody used was mouse α-IgG horseradish peroxidase ( Aliquots from each collected sample were analyzed by Western blot as described in Example 2, except at a 1: 20,000 dilution of Pierce Biotechnology, Rockford, IL). Using antibody-containing sera derived from 3 mice (mouse 2, mouse 3 and mouse 4), a single band corresponding to the SNAP-25 ₁₉₇ cleavage product was detected in the BoNT / A treated sample. Was not detected in the untreated control. Thus, according to the cell-based cleavage assay, of the mice used for immunization, 3 mice (mouse 2, mouse 3 and mouse 4) had higher titers and the P ₁ residue of the BoNT / A cleavage site cleavable bond. Had additional specificity for the SNAP-25 antigen with a carboxyl terminus.

3. Create a hybridoma producing an alpha-SNAP-25 monoclonal antibody that can selectively bind to a SNAP-25 antigen having a generation BoNT / A cleavage site scissile bond carboxyl-terminus at the _{P 1} residue of the hybridoma To this end, spleens were collected from mouse 2 on day 3 following the final “booster” immunization and spleen cells were fused with myeloma cells P3-X63 Ag8.653 using a standard hybridoma protocol. These cells were seeded in five 96-well plates and hybrids were selected using HAT medium. Comparative ELISA with BirA-HisTag®-SNAP-25 _134-197 and BirA-HisTag®-SNAP-25 _134-206 peptides coated on two separate plates within 8-14 days after fusion An initial screening of approximately 480 parental clones was performed using. A comparative ELISA provided a rapid screening method to identify hybridomas that produce antibodies specific for cleaved SNAP-25 ₁₉₇ . The top 18 clones were subjected to further screening using the cell-based cleavage assay described above and immunostaining of LC / A transfected cells (Table 7).

Conditioned media generated by clones 1D3, 1G10, 2E2, 3C1, 3C3 and 3E8 have a ratio _197/206 of at least 4: 1 for SNAP-25 ₁₉₇ cleavage product relative to SNAP-25 ₂₀₆ uncleaved substrate. SNAP-25 ₁₉₇ -cleavage using α-SNAP-25 antibody with preferential binding specificity and using cell-based cleavage assay and immunostaining of PC12 cells transfected with GFP-LC / A Since the product was detected, these clones were further cloned by limiting dilution. Conditioned medium generated by similar to clones 2C9,2F11,3G2,4D1 and 4G6 is at least 1.5 with respect to _{SNAP-25 206} uncleaved substrate relative to the _{SNAP-25 197} cleavage product: 1 ratio _{206 /} These clones were limited because they included an α-SNAP-25 antibody with a preferential binding specificity of ₁₉₇ and detected SNAP-25 ₂₀₆ -uncleaved product using a cell-based cleavage assay. Further cloning by dilution. These single cell-derived clones were rescreened using comparative ELISA, cell-based cleavage and immunostaining to confirm their affinity and specificity, and antibodies were isotyped using standard procedures. . From clones 1D3B8 (IgM.k), 1G10A12 (IgG3.k), 2C9B10 (IgG3.k), 2E2A6 (IgG3.k), 2F11B6 (IgM.k), 3C1A5 (IgG2a.k) and 3C3E2 (IgG2a.k) Ascites was generated. Clone 3E8 stopped antibody production during the cloning process and could not be further evaluated.

4. can be selectively coupled to the SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the alpha-SNAP-25 binding specificity of monoclonal antibodies evaluated BoNT / A cleavage site scissile bond alpha-SNAP To assess the binding specificity of the 25 monoclonal antibody, using ascites from clones 1D3B8, 1G10A12, 2C9B10, 2E2A6, 2F11B6, 3C1A5 and 3C3E2, using cell-based activity assays, immunocytochemistry and immunoprecipitation SNAP-25 cleavage product was detected.

For cell-based activity assays, binding specificity was determined by analyzing the ability of α-SNAP-25 antibody-containing ascites to detect uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage products by Western blot analysis. . Appropriate density of PC12 cells are plated on 60 mm ² tissue culture plates containing 3 mL of appropriate serum medium, grown under 5% carbon dioxide in an incubator at 37 ° C. until reaching the appropriate cell density, and as described above. A transfection solution lacking the pQBI-25 / GFP-BoNT / A-LC expression construct (untransfected cells), or a transfection containing the pQBI-25 / GFP-BoNT / A-LC expression construct Transfected with either solution (transfected cells). Cells were washed and harvested as described in Example 1. To detect the presence of both uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage product, the primary antibody used was a 1: 100 dilution of ascites containing α-SNAP-25 monoclonal antibody and was used Each was harvested as described in Example 1 except that the secondary antibody was a 1: 20,000 dilution of α-mouse IgG (Pierce Biotechnology, Rockford, IL) conjugated to horseradish peroxidase. An aliquot from the sample was analyzed by Western blot. In addition, three commercially available mouse α-SNAP-25 monoclonal antibodies were tested. α-SNAP-25 antibody, SMI-81 (Sternberger Monoclonals Inc., Lutherville, MD), shown by the manufacturer to detect both uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage product Were used at 15,000 dilution according to the manufacturer's recommendations. The α-SNAP-25 antibody MC-6050 (Research & Diagnostic Antibodies, Las Vegas, NV) (shown by the manufacturer to detect both uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage product) Were used at a 1: 100 dilution according to the manufacturer's recommendations. The α-SNAP-25 antibody MC-6053 (Research & Diagnostic Antibodies, Las Vegas, NV), which has been shown by the manufacturer to detect only SNAP-25 ₁₉₇ cleavage products, according to the manufacturer's recommendations Used at 1: 100 dilution.

Table 8 shows α-SNAP-25 antibody-containing ascites in which only the SNAP-25 ₁₉₇ cleavage product was detected. Ascites generated from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 by cell-based cleavage assay synthesized α-SNAP-25 monoclonal antibody with high binding specificity for SNAP-25 ₁₉₇ cleavage product, and SNAP -25 ₂₀₆ relative to the uncleaved substrate permits selective recognition of this cleavage product. The commercially available antibody SMI-81 detected SNAP-25 ₂₀₆ uncleaved substrate, but recognized little SNAP-25 ₁₉₇ cleavage product (Table 8). Surprisingly, the commercially available antibody MC-6050 detected only SNAP-25 ₂₀₆ uncleaved substrate and failed to recognize the SNAP-25 ₁₉₇ cleavage product (Table 8). Even more surprisingly, despite the fact that the commercially available antibody MC-6050 detects only SNAP-25 ₂₀₆ uncleaved substrate and fails to recognize the SNAP-25 ₁₉₇ cleavage product, the manufacturer Advertises that it specifically detects the SNAP-25 ₁₉₇ cleavage product (Table 8). Thus, this analysis shows that 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 exhibit adequate selectivity for the SNAP-25 ₁₉₇ cleavage product, while 1G10A12 and 2F11B6 do not. In addition, the commercially available antibodies SMI-81, MC-6050, and MC-6053 all failed to selectively detect SNAP-25 ₁₉₇ cleavage products and were all unsuitable for the immuno-based methods disclosed in this application. It is.

For immunocytochemistry analysis, binding specificity was determined by analyzing the ability of ascites containing α-SNAP-25 antibody to detect uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage product by immunostaining. . See, for example, “Cell membrane localization signals in the light chain of botulinum neurotoxin” Ester Fernandez-Salas et al., Proc. Natl. Acad. Sci., USA 101 (9): 3208-3213 (2004). PC12 cells of appropriate density are seeded and grown as described above and transfection solution lacking the pQBI-25 / GFP-BoNT / A-LC expression construct (untransfected cells) or pQBI-25 / Transfected with either of the transfection solutions (transfected cells) containing the GFP-BoNT / A-LC expression construct. Cells were washed with 1 × PBS and fixed in 5 mL of PAF for 30 minutes at room temperature. The fixed cells are washed in phosphate buffered saline, incubated in 5 mL of 0.5% Triton® X-100 (polyethylene glycol octylphenol ether) in 1 × PBS and washed with 1 × PBS. And permeabilized in 5 mL of methanol at −20 ° C. for 6 minutes. Permeabilized cells were blocked in 5 mL of 100 mM glycine for 30 minutes at room temperature, washed in 1 × PBS, and blocked in 5 mL of 0.5% BSA in 1 × PBS for 30 minutes at room temperature. Blocked cells were washed in 1 × PBS and incubated for 2 hours at room temperature in 0.5% BSA in 1 × PBS containing a 1:10 dilution of ascites from the clonal hybridoma cell line under test. . Cells probed with primary antibody were washed 3 times in 1 × PBS for 5 minutes each time. 1: 200 of goat polyclonal anti-mouse immunoglobulin G, heavy and light chain (IgG, H + L) antibodies conjugated to ALEXA® FLUOR568 (Invitrogen Inc., Carlsbad, Calif.) As the secondary antibody for the washed cells. Incubated for 2 hours at room temperature in 1 × PBS containing dilution. Cells probed with secondary antibody were washed 3 times for 5 minutes each time in 1 × PBS. Washed cells were prepared for microscopic examination by mounting with VECTASHIELD® mounting medium (Vector Laboratories, Burlingame, Calif.) And mounting a coverslip. An image of signal detection was obtained with a Leica confocal microscope using the appropriate laser settings. Table 8 shows ascites containing α-SNAP-25 antibody that specifically detects SNAP-25 ₁₉₇ -cleavage products. By immunocytochemistry analysis, ascites generated from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 synthesized an α-SNAP-25 monoclonal antibody with high binding specificity for the SNAP-25 ₁₉₇ cleavage product, and SNAP- It has been shown to allow preferential recognition of this cleavage product relative to 25 ₂₀₆ uncut substrate.

For immunoprecipitation analysis, Protein A (HiTrap ™ Protein A HP column, GE Healthcare, Amersham, Piscataway, NJ) purified α-SNAP-25 monoclonal antibody was uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25. Binding specificity was determined by analyzing the ability to precipitate the ₁₉₇ cleavage product. See, eg, Chapter 8 Storing and Purifying Antibodies, pp. 309-311, Harlow & Lane, supra, 1998a. PC12 cells of appropriate density are seeded and grown as described above, and a transfection solution containing the pQBI-25 / GFP expression construct (control cells; SEQ ID NO: 53), or pQBI-25 / GFP-BoNT / Transfected with any of the transfection solutions containing the A-LC expression construct (experimental cells). The pQBI-25 / GFP expression construct comprises an expression vector whose promoter element is operably linked to a polynucleotide encoding GFP of SEQ ID NO: 54. After overnight incubation, the cells were washed by aspirating the growth medium and rinsing each well with 200 μL of 1 × PBS. To harvest the cells, was aspirated PBS, 50mM HEPES, 150mM NaCl, 1.5mM MgCl 2, 1mM EGDT, 10% glycerol, 1% Triton (TM) X-100 (polyethylene glycol octylphenol ether) and 1 × Cells were lysed by adding immunoprecipitation lysis buffer comprising COMPLETE ™ protease inhibitor cocktail (Roche Applied Biosciences, Indianapolis, IN) and incubating at 4 ° C for 1 hour. Lysed cells were centrifuged at 3000 × g for 10 minutes at 4 ° C. to remove cell debris and the supernatant was transferred to a clean tube and diluted to a protein concentration of approximately 1 mg / mL. Approximately 5 μg of purified monoclonal antibody was added to 0.5 mL of diluted supernatant and incubated at 4 ° C. for 2 hours. Following the primary antibody incubation, approximately 50 μL of immobilized protein G (Pierce Biotechnology, Rockford, IL) was added to the diluted supernatant and incubated at 4 ° C. for 1 hour. Add 0.5 mL of immunoprecipitation lysis buffer, centrifuge at 300 × g for 1 minute at 4 ° C. to pellet the immobilized protein G, and decant the supernatant for 3 minutes each time for 30 minutes. Washed twice. After washing, the pellet was resuspended in 30 μL of 1 × SDS loading buffer and the sample was heated to 95 ° C. for 5 minutes. To detect the presence of both uncleaved SNAP-25 ₂₀₆ substrate and SNAP-25 ₁₉₇ cleavage product, the primary antibody used was 1: 1 of α-SNAP-25 polyclonal antibody serum (see Example 4), As described in Example 1 except that the secondary antibody used was a 1: 20,000 dilution of rabbit α-IgG horseradish peroxidase (Pierce Biotechnology, Rockford, IL). An aliquot from each collected sample was analyzed by Western blot. Table 8 shows ascites containing α-SNAP-25 antibody that specifically reduces SNAP-25 ₁₉₇ -cleavage products by immunoprecipitation analysis. By immunoprecipitation analysis, ascites produced from clones 2E2A6 and 3C1A5 synthesized an α-SNAP-25 monoclonal antibody with high binding specificity for the SNAP-25 ₁₉₇ cleavage product, relative to SNAP-25 ₂₀₆ uncleaved substrate. It has been shown to allow preferential recognition of the cleavage product.

5. Evaluation of Binding Affinity of α-SNAP-25 Monoclonal Antibody The binding affinity of α-SNAP-25 monoclonal antibody showing high binding specificity for either SNAP-25 ₁₉₇ cleavage product or SNAP-25 ₂₀₆ uncleaved substrate. To determine, a binding affinity assay was performed using a carboxymethyldextran (CM5) sensor chip (BIAcore, Inc., Piscataway, NJ) on a BIAcore 3000 instrument. A HBS-EP buffer comprising 10 mM HEPES (pH 7.4), 150 mM sodium chloride, 3 mM EDTA, 0.005% (volume / volume) surfactant P20 was run at 25 ° C. and a flow rate of 10 μL / min. SEQ ID NO: 5 amino acids _{134-197 (SNAP-25} 134-197) or SEQ ID NO: 5 amino acids 134-206 standard amine coupling the SNAP-25 peptide comprising the _{(SNAP-25 134-206)} And attached to the surface of the CM5 sensor chip by covalent bonding. Briefly, a CM5 chip was activated by a 7 minute injection of a mixture of 0.2M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.05M N-hydroxysuccinimide; then the SNAP-25 peptide was Injected in 10 mM sodium acetate (pH 4.0) for 20 minutes at a flow rate of 10 μL / min; and unreacted succinimide ester was blocked by a 7-minute injection of 1M ethanolamine hydrochloride (pH 8.5). The amount of SNAP-25 _134-197 or SNAP-25 _134-206 immobilized on the chip was reflected by a 100-150 increase in response units (approximately 0.10-0.15 ng / mm ² ). An antibody sample comprising either ascites generated from clones 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 or a purified monoclonal antibody, as well as a commercially available α-SNAP-25 antibody, were placed on the surface of the CM5 chip. Allowed an association time of 10 minutes and a dissociation time of 20 minutes. The surface was regenerated during the run with a 1 minute injection of 10 mM glycine-HCl (pH 2.5) at a flow rate of 15 μL / min. The sensorgram curve was fitted to a 1: 1 kinetic binding model using BIAevaluation 3.0 software.

The results show that both 2E2A6 and 3C1A5 were highly specific for the SNAP-25 ₁₉₇ cleavage product over the SNAP-25 uncleaved substrate (Table 9). When compared to the binding affinities of MC-6050 and MC-6053, 1D3B6 had an equilibrium dissociation constant approximately 10-fold higher for the SNAP-25 cleavage product relative to these commercially available antibodies (Table 9). Interestingly, only 2E2A6 had a slightly lower equilibrium dissociation constant for the SNAP-25 cleavage product relative to these commercially available antibodies (0.405 nM vs. 0.497 and 0.508) (Table 9). None of these commercially available α-SNAP-25 antibodies selectively recognized SNAP-25 cleavage products (Table 8), but an equilibrium dissociation constant lower than about 0.5 nM achieves such selectivity. Seems to be essential to some extent. Similarly, when compared to the binding affinities of MC-6050 and MC-6053, 2E2A6 is about at least a 1-fold slower off rate / dissociation constant (6.74 × 10 ⁻⁵ vs. 8.82 × 10 ^{− 4} s ⁻¹ and 1.18 × 10 ⁻³ s ⁻¹ ) (Table 9). This further suggests that a separation rate / dissociation constant slower than about 8.82 × 10 ⁻⁴ appears to be somewhat essential to achieve selective binding for the SNAP-25 cleavage product in part. This result is consistent with 1D3B8 having a separation rate / dissociation constant of 5.78 × 10 ⁻⁵ s ⁻¹ (Table 9).

6. can be selectively coupled to the SNAP-25 antigen having a carboxyl-terminus at _{the P 1} residue of sequencing the BoNT / A cleavage site scissile bond epitope from isolated alpha-SNAP-25 monoclonal antibodies In order to determine the epitope of the isolated α-SNAP-25 monoclonal antibody, the variable heavy (V _H ) and variable of the α-SNAP-25 monoclonal antibody produced by the hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 Polynucleotide molecules encoding the light (V _L ) strand were sequenced. MRNA was extracted and purified from each hybridoma using standard protocols and reverse transcribed into cDNA using either oligo dT antisense primers or gene specific (murine IgG1 CH and kappa CL) antisense primers. . After cDNA generation, the cDNA was amplified by PCR using specific murine and human constant domain primers to determine the antibody isotype. Variable domains were amplified from cDNA using _VH and _VL degenerate primers. For 5 'RACE, a homopolymer dCTP tail was added to the 3' end of the cDNA. The heavy and light chains were then amplified using oligo dG sense primers and gene specific (CH / KC) antisense primers. The PCR product contained signal peptide, variable domain and constant domain sequences up to the antisense primer. The PCR product was gel purified to remove small fragments and cloned into blunt or TA vectors for sequencing. Five independent clones were sequenced for each chain, and V _H and V _L chain alignment and consensus sequences were determined (Table 10). The methods used to determine V _H and V _L amino acid sequences are, for example, “New Bioactive Lipid Derivatives and Methods for Making and Using the Same,” Roger A. Sabbadini et al., US Patent Application Publication No. 2007/0281320; And "Molecular characterization of a murine humoral immune response against a botulinum neurotoxin type A binding domain as assessed by using a phage antibody library" Peter Amersdorfer, et al., Infect. Immun. 65 (9): 3743-3752, each of which is hereby incorporated by reference in its entirety. In addition, commercial services are available to sequence the variable heavy (V _H ) and variable light (V _L ) chains of antibodies and identify CDR regions, see, eg, Fusion Antibodies Ltd., Northern Ireland .

The polynucleotide sequence comprising the V _H and V _L chains of the α-SNAP-25 monoclonal antibody produced by the hybridoma disclosed herein is as follows: 1D3B8V _H (SEQ ID NO: 71), 2C9B10V _H (SEQ ID NO: 73), 2E2A6V _H (SEQ ID NO: 75), 3C1A5V _H variant 1 (SEQ ID NO: 77), 3C1A5V _H variant 2 (SEQ ID NO: 79), 3C3E2V _H (SEQ ID NO: 81); 1D3B8V _L (SEQ ID NO: 83), 2C9B10V _L (SEQ ID NO: 85), 2E2A6V _L (SEQ ID NO: 87), 3C1A5V _L (SEQ ID NO: 89) and 3C3E2V _L (SEQ ID NO: 91). The amino acid sequence comprising the V _H and V _L chains of the α-SNAP-25 monoclonal antibody produced by the hybridoma disclosed herein is as follows: 1D3B8V _H (SEQ ID NO: 72), 2C9B10V _H (SEQ ID NO: 74), 2E2A6V _H (SEQ ID NO: 76), 3C1A5V _H variant 1 (SEQ ID NO: 78), 3C1A5V _H variant 2 (SEQ ID NO: 80), 3C3E2V _H (SEQ ID NO: 82); 1D3B8V _L ( SEQ ID NO: 84), 2C9B10V _L (SEQ ID NO: 86), 2E2A6V _L (SEQ ID NO: 88), 3C1A5V _L (SEQ ID NO: 90) and 3C3E2V _L (SEQ ID NO: 92). The amino acid sequence comprising the V _H and V _L CDR domains of the α-SNAP-25 monoclonal antibody produced by hybridomas 1D3B8, 2C9B10, 2E2A6, 3C1A5 and 3C3E2 is shown in Table 10.

Non-limiting examples of amino acid sequences comprising V _H CDR domain variants of the α-SNAP-25 monoclonal antibody produced by the hybridomas disclosed herein include V _H CDR1 variant SEQ ID NO: 118 for 1D3B8; V _H CDR1 variant SEQ ID NO: 119 for 2C9B10, 2E2A6 and 3C1A5V _H variant 2; V _H CDR1 variant SEQ ID NO: 120 for 3C1A5V _H variant 1 and 3C3E2; V _H CDR2 variant SEQ ID NO: 121 for 1D3B8 and 2E2A6; 2C9B10 and 3C1A5V regard _H variant 2 _V H CDR2 variant SEQ ID NO: 122; _V H CDR2 barrier respect 3C1A5V _H variant 1 and 3C3E2 Preparative SEQ ID NO: 123; 1D3B8 and _V H CDR3 variants MDY respect 2C9B10; 2E2A6 and 3C1A5V _V with respect to _H Variant 2 H CDR3 variant MGY; and 3C1A5V _H with respect to variants 1 and 3C3E2 _V H CDR3 variants SEQ ID NO: 124 is included It is. Non-limiting examples of amino acid sequences comprising V _L CDR domain variants of α-SNAP-25 monoclonal antibodies produced by the hybridomas disclosed herein include V _L CDR1 variant SEQ ID NO: 125 for 1D3B8; _V L CDRl variants SEQ ID NO respect 2C9B10: 126; with respect 2E2A6 _V L CDRl variant SEQ ID NO: 127; with respect 3C1A5 _V L CDR1 variant SEQ ID NO: 128; with respect 3C3E2 _V L CDR1 variant SEQ ID NO: 129; for 1D3B8 V _L CDR2 variant KVS; V _L CDR2 variant NAK for 2C9B10; V _L CDR2 variant LVS for 2E2A6; V _L CDR2 variant Y for 3C1A5 For AT; and 3C3E2, the _VL CDR2 variant YAS is included.

BoNT / A to the SNAP-25 epitope having a free carboxyl-terminus at the _{P 1} residue of the cleavage site scissile bond following examples the development of alpha-SNAP-25 polyclonal antibodies that selectively bind the BoNT / A cleavage site cleavable Illustrates how to make an α-SNAP-25 polyclonal antibody capable of selectively binding to a SNAP-25 epitope having a carboxyl terminus at the binding P ₁ residue.
To develop SNAP-25 α-SNAP-25 polyclonal antibodies that bind to _{the P 1} residue epitope comprising a carboxyl-terminus at from the BoNT / A cleavage site scissile bond from a 10- residue The peptide CGGGRIDEANQ (SEQ ID NO: 46) was designed as a SNAP-25 cleavage product antigen. This peptide comprises an N-terminal cysteine residue for conjugation to KLH, a G spacer (GGG) which is a mobile spacer linked to amino acids 191-197 of human SNAP-25 (SEQ ID NO: 5), and It has a carboxylated C-terminal glutamine. A Blast search showed that this peptide has high homology only to SNAP-25 and has little potential for cross-reactivity with other proteins in neuronal cells. The sequence was also carefully scrutinized using computer algorithms to determine the hydrophobic hydrophilicity index, protein surface probability, mobility region and preferred secondary structure, followed by the appropriate orientation and presentation of the selected peptide sequence. . Peptides were synthesized and conjugated to keyhole limpet hemocyanin (KLH) to increase immunogenicity. To identify animals that were not immunoreactive with the proteins present in the cell lysate before immunizing the animals, naïve rabbits were first subjected to western blotting against cell lysates from candidate cell lines. Screened. Two pre-screened rabbits were immunized with this peptide, and after about 3 immunizations in about 8 weeks, the rabbits were bled for testing. The blood was allowed to clot by incubating at 4 ° C. for 60 minutes. The coagulated blood was centrifuged at 10,000 × g for 10 minutes at 4 ° C. to pellet cell debris. The resulting serum sample was aliquoted into 50 μL aliquots and stored at −20 ° C. until needed.

Using a similar scheme based on other SNAP-25 antigens disclosed herein, including the carboxyl terminus at the P ₁ residue from the BoNT / A cleavage site cleavable bond from the SNAP-25 cleavage product. The α-SNAP-25 polyclonal antibody that binds to the epitope is developed. For example, instead of the SNAP-25 antigen of SEQ ID NO: 46, the SNAP-25 antigen of SEQ ID NO: 47 can be conjugated to KLH. As another illustration, amino acids 191-197 of human SNAP-25 from the SNAP-25 antigen of SEQ ID NO: 38 are represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: : 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44, SEQ ID NO: 147 or SEQ ID NO: 148.

2. α-SNAP-25 SNAP- 25 antigen selectively bind alpha-SNAP-25 polyclonal capable of having a carboxyl-terminus at _{the P 1} residue of the screening BoNT / A cleavage site scissile bond for the presence of polyclonal antibodies To determine the presence of antibodies, comparative ELISA and cell-based cleavage assays were performed using rabbit serum extracted as described in Example 3. Sera from both rabbits contained alpha-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond. α-SNAP-25 rabbit polyclonal antibodies were designated NTP22 and NTP23.

3. alpha-SNAP-25 polyclonal antibody purified BoNT / A cleavage site scissile bond alpha-SNAP-25 polyclonal antibodies that can selectively bind to a SNAP-25 antigen having a carboxyl-terminus at _{the P 1} residue of For purification, NTP22 and NTP23 antibodies were purified from rabbit serum using an affinity column containing the SNAP-25 antigen of SEQ ID NO: 46.

4. can be selectively coupled to the SNAP-25 antigen having a carboxyl-terminus at the _{P 1} residue of the alpha-SNAP-25 binding specificity of a polyclonal antibody evaluation BoNT / A cleavage site scissile bond alpha-SNAP To assess the binding specificity of 25 polyclonal antibodies, NTP22 and NTP23 α-SNAP-25 polyclonal antibodies purified using cell-based activity assays, immunocytochemistry and immunoprecipitation as described in Example 3 Was used to detect cleavage products. Cell-based cleavage assays, immunocytochemical analysis and immunoprecipitation analysis all showed that NTP22 and NTP23 α-SNAP-25 polyclonal antibodies did not cross-react with uncleaved SNAP-25. Thus, both NTP22 and NTP23 have a high binding specificity for the SNAP-25 ₁₉₇ cleavage product, allowing preferential recognition of this cleavage product relative to the SNAP-25 ₂₀₆ uncleaved substrate. SPR can be used in BiAcore to determine affinity for antigen as described in Example 3.

Example of components and conditions prepared following for sandwich ELISA is using specific alpha-SNAP-25 monoclonal antibody to SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond How to identify and prepare the components and conditions necessary to perform a sandwich ELISA useful for performing an immune-based method of detecting BoNT / A activity by detecting SNAP-25 cleavage products Illustrate.
1. Preparation of cell lysate from cells treated with BoNT / A To obtain a BoNT / A-treated cell lysate for analysis, cells of the appropriate density from a stock culture of Neuro-2a are added to the minimum essential medium. Inoculated into T175 flasks containing 50 mL of serum-free medium containing 2 mM GlutaMAX ™ I, 1 × B27 supplement, 1 × N2 supplement, 0.1 mM non-essential amino acids, 10 mM HEPES with Earl's salt. These cells are differentiated under approximately 5% carbon dioxide in a 37 ° C. incubator until the cells are differentiated as assessed by standard and routine morphological criteria such as growth inhibition and neurite outgrowth (approximately 2 to 3 Day). As a control, cells of appropriate density from a Neuro-2a stock culture were seeded in T175 flasks containing 50 mL of the appropriate growth medium (Table 1). These undifferentiated control cells were grown under 5% carbon dioxide in a 37 ° C. incubator until the cells reached 50% confluence (approximately 18 hours). Medium from both differentiated and undifferentiated control cultures was aspirated from each well and replaced with fresh medium containing either 0 (untreated sample) or 10 nM BoNT / A complex. After overnight incubation, cells were washed and at 4 ° C. in freshly prepared Triton X-100 lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl ₂ , 1 mM EGTA, 1% Triton X-100). Cells were harvested by lysis with constant agitation for 30 minutes. Lysed cells were centrifuged at 4000 rpm, 4 ° C. for 20 minutes, and debris was eliminated using bench top centrifugation. Cell lysate protein concentration was measured by Bradford assay.

2. Preparation and identification of sandwich ELISA components To identify appropriate capture antibody-detection antibody pairs, including 11 different α-SNAP-25 capture antibodies and 7 different α-SNAP-25 detection antibodies ECL sandwich ELISA analysis was performed with a total of 26 different combinations of capture and detection antibody pairs (Table 12). The α-SNAP-25 antibody used was the 2E2A6 and 3C1A5 α-SNAP-25 mouse monoclonal antibody disclosed herein, SMI-81, MC-6050 and MC-6053 α-SNAP disclosed herein. -25 mouse monoclonal antibody, NTP23 α-SNAP-25 rabbit polyclonal antibody disclosed herein, S9684 α-SNAP-25 rabbit polyclonal antibody (Sigma, St. Louis, MO), H-50 α-SNAP-25 Rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), C-18 α-SNAP-25 goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), N-19 α-SNAP-25 Goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and SP 12 α-SNAP-25 mouse polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).

  To prepare a capture antibody solution, α-SNAP-25 monoclonal antibody contained in ascites from hybridoma cell lines 2E2A6 and 3C1A5, and α-SNAP-25 MC-6050 and MC-6053 monoclonal antibodies were added to standard proteins. Purified using A purification protocol. All other α-SNAP-25 antibodies were purchased as purified antibodies.

  To prepare a detection antibody solution, the appropriate α-SNAP-25 antibody was prepared according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, MD) ruthenium (II) -tris-bipyridine- (4-methylsulfonate) NHS. Conjugated to an ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD). Conjugation reaction was performed by adding 30 μL of MSD SULFO-TAG ™ stock solution reconstituted in distilled water to 200 μL of 2 mg / mL α-SNAP-25 polyclonal antibody and incubating the reaction at room temperature in the dark for 2 hours. did. The labeled antibody was purified using a standard spin column protocol and the protein concentration determined using a standard colorimetric protein assay. The absorbance of the α-SNAP-25 antibody / MSD SULFO-TAG ™ conjugate was measured at 455 nm using a spectrophotometer to determine the molar concentration per liter. The detection antibody solution was stored at 4 ° C. until needed.

  To prepare a solid support comprising a capture antibody that is specific for the SNAP-25 cleavage product, add approximately 5 μL of an appropriate α-SNAP-25 monoclonal antibody solution (20 μg / mL in 1 × PBS) to 96 Add to each well of a well MSD High Bind plate and allow to air dry in a biological safety cabinet for 2-3 hours to evaporate the solution. The capture antibody binding wells were then added by adding 150 μL of blocking buffer containing 2% Amersham blocking reagent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA) for 2 hours at room temperature. Shut off. The blocked plate was sealed and stored at 4 ° C. until needed.

  To detect the presence of the SNAP-25 cleavage product cleaved by ECL sandwich ELISA analysis, blocking buffer from the stored plate was aspirated from the wells and from BoNT / A treated cells as described above. 25 μL of the lysate was added to each well and the plate was incubated at 4 ° C. overnight. Plate wells 3 times by aspirating cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Was washed. After washing, 25 μL each of 5 μg / mL detection antibody solution containing 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Added to the wells, the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After incubating the detection antibody, the wells were washed 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 150 μL of 1 × Read Buffer (Meso Scale Discovery, Gaithersburg, MD) is added to each well and using a SECTOR ™ Imager 6000 image reader (Meso Scale Discovery, Gaithersburg, MD). The plate was read. Ratios were calculated by dividing the signal obtained at the 10 nM dose for each antibody pair by the signal obtained at the 0 nM dose for each antibody pair (Table 12). These results indicate that, of the 26 different combinations of antibody pairs tested, only 3 antibody pairs had a signal-to-noise ratio exceeding 10: 1 for the high dose tested: first pair (2E2A6 Mouse mAb and S9684 rabbit pAb), 4th pair (3C1A5 mouse mAb and S9684 rabbit pAb) and 18th pair (S9684 rabbit pAb and 2E2A6 mouse mAb). The first antibody pair was chosen for further assay development.

3. Optimization of cell differentiation conditions To determine optimal differentiation conditions for cell lines comprising cells sensitive to BoNT / A intoxication when using a sandwich ELISA detection system, various cell culture media and differentiation times are used. Both lengths were tested.
To determine the optimal differentiation medium, the appropriate density of cells from the SiMa cell line is divided into the following medium and differentiation supplements: 1) RPMI 1640, 10% fetal calf serum, 1% penicillin-streptomycin, 2 mM L-glutamine and 25 μg. / ML GT1b); 2) RPMI-1640, 1 × B27 supplement, 1 × N2 supplement and 25 μg / mL GT1b; 3) Minimum essential medium, 1 × B27 supplement, 1 × N2 supplement and 25 μg / mL GT1b; and 4) Seed wells of a 24-well cell culture plate coated with type IV collagen containing 1 mL of RPMI-1640, 10% BSA, 1 × N2 supplement, 1 × NGF supplement and 25 μg / mL GT1b; . Cells were incubated in 5% carbon dioxide in an incubator at 37 ° C. until cells differentiated (approximately 3 days) as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. . Medium was aspirated from each well and replaced with fresh medium containing either 0 (untreated sample), 0.2 pM, 2 pM or 20 pM BoNT / A complex. After overnight treatment, the cells were washed and incubated for an additional 2 days without toxin to allow cleavage of the SNAP-25 substrate and harvested as described above in Section 1. Cell lysate protein concentration was measured by Bradford assay. Detection of the presence of SNAP-25 cleavage product by ECL sandwich ELISA analysis was performed as described above using the first antibody pair. As discussed in Example 1, undifferentiated cells did not take up toxins as effectively as differentiated cells. The most effective differentiation medium to increase BoNT / A uptake and consequently SNAP-25 cleavage is medium 3 (MEM + N2 + B27) followed by medium 2 (RPMI-1640 + N2 + B27), and medium 4 (RPMI-1640 + N2 + NGF + BSA) (FIG. 3). Cells cultured in medium 2 resulted in further cleavage of SNAP-25 compared to other media.

In order to determine the optimal differentiation time, cells of appropriate density from the SiMa cell line are added to the minimum essential medium, 2 mM GlutaMAX ™ I, 1 × B27 supplement, 1 × N2 supplement, 0.1 mM non-essential with Earl's salt. The wells of a 96-well cell culture plate coated with poly-D-lysine containing 100 μL of serum-free medium containing essential amino acids, 10 mM HEPES and 25 μg / mL GT1b were plated. Cells were seeded on 4 different days to obtain differentiation time courses tested at 6, 24, 48 and 72 hours and incubated under 5% carbon dioxide in a 37 ° C. incubator. Medium is aspirated from each well and 0 (untreated sample), 0.1 pM, 0.2 pM, 0.4 pM, 0.8 pM, 1.6 pM, 3.1 pM, 6.25 pM, 12.5 pM or 25 pM BoNT Replaced with fresh medium containing either of the / A complexes. After overnight treatment, the cells were washed and incubated for an additional 2 days without toxin to allow cleavage of the SNAP-25 substrate and harvested as described above in Section 1. After harvesting, detection of the presence of SNAP-25 cleavage products by cell lysate protein concentration and ECL sandwich ELISA analysis was performed as described above. The raw data obtained from the ECL imager was then transferred to SigmaPlot v. 9.0 and a dose-response curve was defined using a four parameter logistic fit. When plotting the data, there were no constraints used for the four parameter logistic function. The following analysis was used to generate a graph report: R2 (correlation coefficient), a (maximum for the data set), b (slope) and X0 ± SE (EC ₅₀ values ± standard error). Results showed that EC ₅₀ values of less than 2 pM could be achieved with cells differentiated for 48-72 hours (FIG. 4). The finding that differentiated cells between 48 and 72 hours of differentiation could be used without significant changes in cell performance highlighted the robustness of the assay. Although differentiation times of less than 48 hours may not be appropriate for testing picomolar concentrations of formulated products, these short differentiation times are sensitive enough for bulk drug substance testing.

4. Optimization of BoNT / A treatment time Various lengths of BoNT / A treatment time were tested to determine the optimal length of time for cells from cell lines that need to be treated with BoNT / A. did. Appropriate density of cells from the SiMa cell line was obtained using minimal essential medium, 2 mM GlutaMAX ™ I, 1 × B27 supplement with Earl's salt, 1 × N2 supplement, 0.1 mM non-essential amino acid, 10 mM HEPES and 25 μg / mL GT1b Seeded into wells of a 96-well cell culture plate coated with poly-D-lysine containing 100 μL of serum-free medium containing Cells are seeded and cultured under approximately 5% carbon dioxide in an incubator at 37 ° C. until cells differentiate (as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth (approximately 3 days). ) Incubated. Medium is aspirated from each well and 0 (untreated sample), 0.1 pM, 0.2 pM, 0.4 pM, 0.8 pM, 1.6 pM, 3.1 pM, 6.3 pM, 12.pM in RPMI 1640 growth medium. Three samples were replaced with fresh medium containing either 5 pM or 25 pM BoNT / A complex to create a full dose-response. Five different length BoNT / A treatment schemes were implemented: 1) BoNT / A 6 hour treatment, cell removal and washing, 18 hour cell incubation without BoNT / A, and section 1 above 2) BoNT / A 24 hr treatment, cell removal and washing, and cell harvest as described above in section 1; 3) BoNT / A 24 hr treatment, cell removal and Wash, incubate cells for 24 hours without BoNT / A, and harvest cells as described above in section 1; 4) BoNT / A incubate for 24 hours, remove and wash cells, with BoNT / A Without incubation for 48 hours, and harvesting of cells as described above in section 1; and 5) BoNT / A 24 hour incubation, cell removal and washing, 72 hour cell incubation without BoNT / A, and harvesting of cells as described above in section 1. After harvesting, cell lysate protein concentration, detection of SNAP-25 cleavage products by ECL sandwich ELISA was performed, and EC ₅₀ was calculated as described above. The results indicate that any BoNT / A treatment tested can achieve EC ₅₀ values of less than 2 pM (FIG. 5). Interestingly, BoNT / A treatment regimes of 24 hours + 24 hours, 24 hours + 48 hours and 24 hours + 73 hours produced essentially the same EC ₅₀ values, 1.0 pM, 1.1 pM and 0.9 pM, respectively. The EC ₅₀ values generated for the 6 hour + 18 hour and 24 hour + 0 hour BoNT / A treatment regimes were 1.7 pM and 1.6 pM, respectively. Although the amount of signal obtained is low, these results detect BoNT / A activity using BoNT / A treatment time between 6 and 24 hours, plus 1 to 3 days post-treatment incubation It is shown that an EC ₅₀ can be generated that is sufficient to do so, and flexibility can be provided in the overall time course of the assay.

5. Sensitivity of immuno-based methods for detecting BoNT / A activity To assess the sensitivity of the immuno-based methods for detecting BoNT / A activity disclosed herein, cells sensitive to BoNT / A intoxication. The timing of BoNT / A uptake by was determined. Appropriate density of cells from the SiMa cell line was obtained using minimal essential medium, 2 mM GlutaMAX ™ I, 1 × B27 supplement with Earl's salt, 1 × N2 supplement, 0.1 mM non-essential amino acid, 10 mM HEPES and 20 μg / mL GT1b Seeded into wells of a 96-well cell culture plate coated with poly-D-lysine containing 100 μL of serum-free medium containing Cells were incubated in 5% carbon dioxide in an incubator at 37 ° C. until cells differentiated (approximately 3 days) as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. . Medium is aspirated from each well, replaced with fresh medium containing 1 nM BoNT / A complex, and BoNT / A treated cells are removed for 0 min (neurotoxin added and then immediately removed), 5 min Incubated at 6 different time points at 10, 20, 30, and 60 minutes. A negative control of medium (0 nM) without BoNT / A was used. Following incubation, cells were washed and harvested as described above in Section 1. Following harvest, protein concentration of cell lysates, detection of SNAP-25 cleavage products by ECL sandwich ELISA was performed, and EC ₅₀ was calculated as described above. The results showed that uptake of BoNT / A by the cells took less than 1 minute before producing significant amounts of SNAP-25 cleavage product above background (FIG. 6).

6. Specificity of immuno-based methods for detecting BoNT / A activity. To evaluate the specificity of the immuno-based methods for detecting BoNT / A activity disclosed herein, partially inactivated. The ability of cells sensitive to BoNT / A poisoning to accurately distinguish BoNT / A by excluding BoNT / A was determined. Appropriate density of cells from the SiMa cell line was obtained using minimal essential medium, 2 mM GlutaMAX ™ I, 1 × B27 supplement with Earl's salt, 1 × N2 supplement, 0.1 mM non-essential amino acid, 10 mM HEPES and 25 μg / mL GT1b Seeded into wells of a 96-well cell culture plate coated with poly-D-lysine containing 100 μL of serum-free medium containing Cells were incubated in 5% carbon dioxide in an incubator at 37 ° C. until cells differentiated (approximately 3 days) as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. . Medium was aspirated from each well and 1) 0 (untreated sample), 0.03 pM, 0.1 pM, 0.31 pM, 0.93 pM, 2.78 pM, 8.33 pM and 25 pM BoNT / A complex; 2 ) 0, 0.14 nM, 0.41 nM, 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM and 100 nM Inactive BoNT / A (iBoNT / A); or 3) 0, 0.14 nM, 0.14 Replaced with fresh medium containing any of 41 nM, 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM and 100 nM LH _N / A fragment. iBoNT / A contains a mutation in the zinc binding domain of the light chain that completely inactivates the metalloprotease activity of the neurotoxin, eg, “Expression and purification of the light chain of botulinum neurotoxin A: Liquing Zhou, et al., Biochemistry 34: 15175-15181 (1995) (all of which are hereby incorporated by reference) Refer to). The LH _N / A fragment lacks a binding domain, but contains an intact translocation domain and a light chain, eg, “Recombinant Toxin Fragment” Clifford C. Shone et al., US Pat. No. 6,461,617 (all by reference). See part of this specification). After 24 hours of treatment, the cells were washed and incubated for an additional 2 days without toxin to allow cleavage of the SNAP-25 substrate and harvested as described above in Section 1. After harvesting, cell lysate protein concentration, detection of SNAP-25 cleavage products by ECL sandwich ELISA was performed, and EC ₅₀ was calculated as described above. The results show that the binding affinity of cells for iBoNT / A and LH _N / A (EC ₅₀ > 100 nM) is at least 60,000 lower than the binding affinity for BoNT / A (EC ₅₀ = 1.6 pM) (FIG. 7). No SNAP-25 cleavage product was detected in cells treated with all concentrations of iBoNT / A tested. A low amount of SNAP-25 cleavage product was detected in cells treated with the highest dose of LH _N / A fragment, but this activity is due to non-specific uptake of this fragment due to the activity of the translocation domain. Thus, according to the results, the immuno-based method of detecting BoNT / A activity disclosed herein whereby BoNT / A cleaves the SNAP-25 substrate by proteolysis and BoNT / A accepts BoNT / A. All involved in the intoxication process, including binding to the body, internalization of the neurotoxin / receptor complex, translocation of the BoNT / A light chain from the intracellular vesicle to the cytoplasm and proteolytic cleavage of SNAP-25 It is shown that the process can be measured.

The following examples immuno-based method of detecting BoNT / A activity using ECL sandwich ELISA are, SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond using ECL sandwich ELISA Illustrates an immune-based method of detecting BoNT / A activity by detecting SNAP-25 cleavage product using an α-SNAP-25 monoclonal antibody specific for the cleavage product.
To prepare lysates from cells treated with BoNT / A, an appropriate density of cells from an established cell line is obtained using 2 mM GlutaMAX ™ I, 1 × B27 supplement with minimal essential medium, Earl's salt, 1 × B27 supplement, XN2 supplement, 0.1 mM non-essential amino acid, 10 mM HEPES and 25 μg / mL GT1b was seeded into wells of a 96-well tissue culture plate containing 100 μL of serum-free medium (see Examples 1 and 2). These cells are differentiated (approximately 3 days) under 5% carbon dioxide in an incubator at 37 ° C. as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. Incubated. Medium from differentiated cells is aspirated from each well and 0 (untreated sample), 0.03 pM, 0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM, 8.3 pM and 25 pM BoNT / A complex Replaced with fresh medium containing any of the body. After 24 hours of treatment, cells were washed and incubated for an additional 2 days without toxin. Cells were harvested as described in Example 5.

  To prepare an α-SNAP-25 capture antibody solution, α-SNAP-25 monoclonal antibody contained in ascites from hybridoma cell line 2E2A6 was purified using a standard protein A purification protocol. To prepare an α-SNAP-25 detection antibody solution, α-SNAP-25 rabbit polyclonal antibody S9684 (Sigma, St. Louis, MO) was prepared according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, MD) ruthenium ( II) Conjugated to -Tris-bipyridine- (4-methylsulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD). Conjugation reaction, purification of labeled α-SNAP-25 antibody, concentration determination and storage were as described in Example 5.

  To prepare a solid support comprising a capture antibody that is specific for the SNAP-25 cleavage product, approximately 5 μL of α-SNAP-25 monoclonal antibody 2E2A6 solution (20 μg / mL in 1 × PBS) in 96 wells. The solution was added to each well of the MSD High Bind plate and allowed to air dry in a biological safety cabinet for 2-3 hours to evaporate the solution. The capture antibody binding well was then blocked and used to directly detect BoNT / A activity.

To detect the presence of SNAP-25 cleavage product by ECL sandwich ELISA analysis, the blocking buffer from the stored plate is aspirated from the wells and 25 μL of lysate from cells treated with BoNT / A is added to each well. And the plates were incubated overnight at 4 ° C. 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Washed twice. After washing, 25 μL each of 5 μg / mL detection antibody solution containing 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Added to the wells, the plate was sealed, and the sealed plate was incubated at room temperature for 1 hour with shaking. After incubating the detection antibody, the wells were washed 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 150 μL of 1 × Read Buffer (Meso Scale Discovery, Gaithersburg, MD) is added to each well and the plate is used with a SECTOR ™ Imager 6000 image reader (Meso Scale Discovery, Gaithersburg, MD) And read. The collected data was analyzed and the EC ₅₀ was calculated as described in Example 5. Representative results are shown in FIG. 8, and these results show that the signal to noise ratio for the lower asymptote is about 15: 1 to about 20: 1 and the signal to noise ratio for the upper asymptote is about 20: 1 to about 500. (range of about 2.0pM about 0.3 pM) of BoNT / a typical 1.0pM is detected _{EC 50} of 1 has been shown.

The following examples immuno-based method of detecting BoNT / A activity using CL sandwich ELISA is specific for the SNAP-25 having a carboxyl-terminus at the _{P 1} residue of the BoNT / A cleavage site scissile bond by CL sandwich ELISA Illustrates an immune-based method of detecting BoNT / A activity by detecting SNAP-25 cleavage products using a novel α-SNAP-25 monoclonal antibody.
Lysates from the cells were treated with BoNT / A and α-SNAP-25 capture antibody solution was prepared as described in Example 6.
To prepare α-SNAP-25 detection antibody solution, α-SNAP-25 polyclonal antibody S9684 (Sigma, St. Louis, MO) was prepared according to the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL). Conjugated to horseradish peroxidase (HRP). Conjugation reaction was performed by adding 500 μL of 1 mg / mL α-SNAP-25 polyclonal antibody to a vial containing lyophilized activated peroxidase, mixing the components, and then adding 10 μL of sodium cyanoborohydride. did. The reaction mixture was incubated for 1 hour at room temperature in a fume hood. After quenching the reaction, the labeled antibody was purified using a standard spin column protocol, and the protein concentration was determined using a standard colorimetric protein assay. The absorbance of the α-SNAP-25 polyclonal antibody / HRP conjugate was measured at 455 nm using a spectrophotometer to determine the molar concentration per liter. The α-SNAP-25 detection antibody solution was stored at 4 ° C. until needed.

  To prepare a solid support comprising an α-SNAP-25 capture antibody that is specific for the SNAP-25 cleavage product, α-SNAP-25 monoclonal antibody 2E2A6 solution (1 mg / mL in 1 × PBS) Approximately 100 μL was added to each well of a 96 well Greiner white plate and the plate was incubated overnight at 4 ° C. and then the excess antibody solution was discarded. The capture antibody binding wells were then added by adding 150 μL of blocking buffer containing 2% Amersham blocking reagent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA) for 1 hour at room temperature. Shut off. The blocking buffer was discarded and the plate was blot dried by tapping on a paper towel and tapping. The capture antibody binding well was then blocked and used to directly detect BoNT / A activity.

To detect the presence of SNAP-25 cleavage product by CL sandwich ELISA analysis, 50 μL of lysate from cells treated with BoNT / A was added to each well, the plate was sealed, and the sealed plate was placed at 500 rpm. Incubated at 4 ° C. for 2-4 hours to overnight on a shaker rotating at. Remove the plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Washed. After washing, 1 mg / mL α-SNAP-25 polyclonal comprising 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) 100 μL of antibody / HRP detection antibody solution was added to each well, the plates were sealed and the sealed plates were incubated for 1 hour at room temperature on a shaker rotating at 650 rpm. After incubating the detection antibody, the wells were washed 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 100 μL of SuperSignal ELISA Pico 1: 1 mixture (Pierce Biotechnology, Inc., Rockford, IL) was added to each well and read at 395 nm using a luminometer (Molecular Devices, Sunnyvale, Calif.). The collected data was analyzed and the EC ₅₀ was calculated as described in Example 5. These results indicate that the BoNT / A representative of EC _{50 with} a signal to noise ratio of about 15: 1 to about 20: 1 for the lower asymptote and a signal to noise ratio of about 20: 1 to about 500: 1 for the upper asymptote. The value 1.0 pM was shown to be detected (ranging from about 0.3 pM to about 2.0 pM).

Immune-based methods of detecting BoNT / A activity using multiplex ECL sandwich ELISA The following examples are specific for α-SNAP-25 monoclonal antibodies specific for SNAP-25 cleavage products and second antibodies for different proteins 2 illustrates a multiplex immune-based method for detecting BoNT / A activity by using pairs to detect SNAP-25 cleavage products.

1. To obtain an untreated cell lysate for the preparation and identification analysis of a capture antibody-detection antibody pair for the second protein, the appropriate density of cells from a stock culture of SiMa cells is 1 × RPMI 1640, 10% Seeded in T175 flasks containing 40 mL of growth medium containing FBS, 0.1 mM non-essential amino acids, 10 mM HEPES, 1 mM sodium pyruvate and 100 units / 100 μg penicillin-streptomycin. These cells were incubated under 5% carbon dioxide in a 37 ° C. incubator until the cells were approximately 70-90% confluent. Cells were washed and at 4 ° C. in freshly prepared Triton X-100 lysis buffer (20 mM Tris pH 7.5, 150 mM sodium chloride, 0.001 M EDTA, 1 mM EGTA and 1% Triton-X-100). Cells were harvested by lysis with constant agitation for approximately 30 minutes. Lysed cells were centrifuged at 3300-3330 × g for 40 minutes at 8 ° C. and debris was excluded using bench top centrifugation.

  To identify an appropriate capture antibody-detection antibody pair for the second protein, ECL sandwich ELISA analysis was performed on 21 different combinations of capture and detection antibody pairs consisting of 5 different proteins (Table 13). . The antibodies used were α-syntaxin 1A-HPC mouse monoclonal antibody S0664 (Sigma, St. Louis, MO), α-GAPDH mouse monoclonal antibody MAB374 (Chemicon, Temecula, CA), α-syntaxin 1 rabbit polyclonal antibody S1172-1 (Sigma, St. Louis, MO), α-GAPDH rabbit polyclonal antibody 2275-PC-1 (R & D Systems, Minneapolis, Minn.), Α-syntaxin 2 rabbit polyclonal antibody S5687 (Sigma, St. Louis, MO), α-human syntaxin 2 mouse monoclonal antibody MAB2936 (R & D Systems, Minneapolis, Minn.), α-mouse syntaxin 2 goat polyclonal antibody AF2568 (Sigma, St. Louis, MO), α-syntaxin 2 rabbit polyclonal antibody AB5596 ( Sigma, St. Louis, MO), α-syntaxin 1 rabbit polyclonal S1172-2 (Sigma, St. Louis, MO), α-h, m, r actin sheep polyclonal antibody AF4000 (R & D Systems, Minneapolis, Minn.), Α-beta actin mouse monoclonal antibody A1978 (Sigma, St. Louis, MO), α-beta mouse polyclonal antibody actin A2228 (Sigma, St. Louis, MO), mouse α-GAPDH mouse monoclonal antibody G8795 (Sigma, St. Louis, MO), α-GAPDH rabbit polyclonal antibody G9595 (Sigma , St. Louis, MO).

  Monoclonal antibodies were purchased as purified antibodies to prepare a second protein capture antibody solution. To prepare a second protein detection antibody solution, the appropriate antibody is prepared according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, MD) ruthenium (II) -tris-bipyridine- (4-methylsulfonate) NHS ester. Conjugated to a labeling reagent (Meso Scale Discovery, Gaithersburg, MD). The conjugation reaction was performed by adding 30 μL of MSD SULFO-TAG ™ stock solution reconstituted in distilled water to 200 μL of 2 mg / mL polyclonal antibody and incubating the reaction at room temperature in the dark for 2 hours. The labeled antibody was purified using a standard spin column protocol and the protein concentration determined using a standard colorimetric protein assay. The absorbance of the antibody / MSD SULFO-TAG ™ conjugate was measured at 455 nm using a spectrophotometer to determine the molar concentration per liter. The detection antibody solution was stored at 4 ° C. until needed.

  To prepare a solid support comprising a capture antibody specific for the SNAP-25 cleavage product, approximately 5 μL of α-SNAP-25 monoclonal antibody 2E2A6 solution (20 μg / mL in 1 × PBS) in 96 wells. Add to each well of MSD High Bind plate and allow to air dry in a biological safety cabinet for 2-3 hours to liquid evaporate and then seal the plate and store at 4 ° C until needed did. Then containing 3% BSA (Pierce, Rockford, IL), 10% goat serum (Rockland Immunochemicals, Gilbertsville, PA) and 0.05% TWEEN-20 Difco 1% nonfat milk (BD BioSciences Franklin Lakes, NJ) The dried capture antibody binding wells were blocked by adding 150 μL of the resulting blocking buffer at room temperature for 1-2 hours.

  To detect the presence of protein by ECL sandwich ELISA analysis, the blocking buffer from the stored plate is aspirated from the wells and 25 μL of lysate from cells treated with BoNT / A as described above is added to each well. And the plates were incubated overnight at 4 ° C. 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Washed twice. After washing, 25 μL of an appropriate second protein detection antibody solution at 5 μg / mL resuspended in blocking buffer as described above is added to each well, the plates are sealed, and the sealed plates are incubated at room temperature. Incubated for about 1 hour with shaking. After incubating the detection antibody, the wells were washed 3 times with 250 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 150 μL of 1 × Read Buffer (Meso Scale Discovery, Gaithersburg, MD) is added to each well and the plate is used with a SECTOR ™ Imager 6000 image reader (Meso Scale Discovery, Gaithersburg, MD) And read. The ratio was calculated by dividing the signal obtained from the untreated cell lysate for each antibody pair by the signal obtained for the lysis buffer control (0 nM dose) for each antibody pair (Table 13). These results indicate that, of the 21 different combinations of antibody pairs tested, only 2 antibody pairs had a signal to noise ratio of greater than 10: 1 for the high dose tested: 16th vs α- GAPDH mouse monoclonal antibody MAB374 and α-GAPDH rabbit polyclonal antibody RDS2275-PC-1; and 21st pair: α-GAPDH mouse monoclonal antibody MAB374 and α-GAPDH rabbit polyclonal antibody G9545. The α-GAPDH mouse monoclonal antibody MAB374 and α-GAPDH rabbit polyclonal antibody G9545 pair were selected as the second protein capture antibody-detection antibody pair for the multiplex ECL sandwich ELISA.

2. To obtain a BoNT / A-treated cell lysate for analysis of immuno-based methods that detect BoNT / A activity using a multiplex ECL sandwich ELISA , cells of appropriate density from a stock culture of the SiMa cell line Poly-D- containing serum-free medium containing 2 mM GlutaMAX ™ I, 1 × B27 supplement, 1 × N2 supplement, 0.1 mM non-essential amino acids, 10 mM HEPES with minimal essential medium, Earl's salt Lysine 96-well plates were seeded. These cells are differentiated (approximately 3 days) under 5% carbon dioxide in an incubator at 37 ° C. as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. Incubated. Medium is aspirated from each well and 0 (untreated sample), 0.67 units / mL, 2.35 units / mL, 8.23 units / mL, 28.82 units / mL, 101 units / mL, 353 Replaced with fresh medium containing either unit / mL BoNT / A complex. After 24 hours of treatment, the cells were washed and incubated for an additional 2 days without toxin. Cells were washed, harvested and processed as described above in section 1.

  An α-SNAP-25 capture antibody solution and an α-SNAP-25 detection antibody solution were prepared as described in Example 7. To prepare an α-GAPDH capture antibody solution, α-GAPDH monoclonal antibody mouse MAB374 (Chemicon, Temecula, Calif.) was prepared as described in Section 1 above. To prepare an α-GAPDH detection antibody solution, α-GAPDH rabbit polyclonal antibody G9545 (Sigma, St. Louis, MO) was prepared according to the manufacturer's instructions (Meso Scale Discovery, Gaithersburg, MD) ruthenium (II) -Tris. -Bipyridine- (4-methylsulfonate) NHS ester labeling reagent (Meso Scale Discovery, Gaithersburg, MD). Conjugation reactions, purification of labeled α-SNAP-25 antibody, concentration determination and storage were as described in Section 1 above.

  To prepare a solid support comprising an α-SNAP-25 capture antibody and an α-GAPDH capture antibody, an α-SNAP-25 capture antibody solution (45 μg / mL in 1 × PBS) approximately 2.5 nL and α -2.5 nL of GAPDH capture antibody solution (120 μg / mL in 1 × PBS) was added to each well of a 96-well MSD High Bind plate in multiplex format using a robotic system. The solution was liquid evaporated by air drying in a biological safety cabinet for at least 2-3 hours. The capture antibody binding wells were then blocked and used to directly detect BoNT / A activity and GAPDH protein.

  To detect the presence of SNAP-25 cleavage product by multiplex ECL sandwich ELISA analysis, blocking buffer from the stored plate was aspirated from the wells and from BoNT / A treated cells as described above. 25 μL of the lysate was added to each well and the plate was incubated at 4 ° C. overnight. 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Washed twice. After washing, 25 μL of 5 μg / mL α-SNAP-25 detection antibody solution and 25 μL of 5 μg / mL α-GAPDH detection antibody as described above are added to each well, the plate is sealed, and the sealed plate is incubated at room temperature. Incubate for about 1 hour with shaking. After incubating the detection antibody, the wells were washed 3 times with 250 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 150 μL of 1 × Read Buffer (Meso Scale Discovery, Gaithersburg, MD) is added to each well and the plate is used with a SECTOR ™ Imager 6000 image reader (Meso Scale Discovery, Gaithersburg, MD) And read. The collected data is analyzed and the relative potency is calculated from the normalized data as described in Example 5 except that PLA 2.0 software (Stegmann Systems, GmbH, Germany) was used.

  As a comparison, detection of SNAP-25 cleavage products was also performed as described in Example 6 using a singleplex ECL sandwich ELISA.

  Results show one outlier that did not fit a dose-response curve, either from SNAP-25 data obtained from a single plex ECL sandwich ELISA or from denormalized SNAP-25 data obtained from a multiplex ECL sandwich ELISA. The sample was shown to be represented. However, normalization of the SNAP-25 data to the GAPDH data showed a good curve fit and the potency approximated the expected value.

Immune-based methods for detecting BoNT / A activity using a multiplex EC sandwich ELISA The following examples are specific for α-SNAP-25 monoclonal antibodies specific for SNAP-25 cleavage products and second antibodies for different proteins 2 illustrates a multiplex immune-based method for detecting BoNT / A activity by using pairs to detect SNAP-25 cleavage products.

  Lysates were prepared from cells treated with BoNT / A as described in Example 6. α-SNAP-25 capture antibody solution, α-SNAP-25 detection antibody solution and α-SNAP-25 solid support were prepared as described in Example 7.

  To prepare an α-GAPDH capture antibody solution, α-GAPDH monoclonal antibody MAB374 (Millipore, Billerica, MA) was purchased as a purified antibody. To prepare an α-GAPDH detection antibody solution, α-GAPDH polyclonal antibody G9545 (Sigma, St. Louis, MO) was prepared according to the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL) horseradish peroxidase (HRP). ). Conjugation reactions, concentration determination and storage were as described in Example 7.

  To prepare a solid support comprising a second capture antibody specific for the second protein, approximately 100 μL of a monoclonal antibody solution comprising 1 μg / ml α-GAPDH monoclonal antibody MAB374 was added to a 96-well Greiner white plate. Was added to each well and incubated overnight at 4 ° C. and then the excess antibody solution was discarded. An α-GAPDH capture antibody was then added by adding 150 μL of blocking buffer comprising 2% Amersham blocking reagent (GE Life Sciences, Piscataway, NJ) and 10% goat serum (VWR, West Chester, PA) for 1 hour at room temperature. Binding wells were blocked. The blocking buffer was discarded and the plate was blot dried by tapping on a paper towel and tapping. The capture antibody binding well was then blocked and used to directly detect BoNT / A activity.

To detect the presence of SNAP-25 cleavage products by multiplex CL sandwich ELISA analysis, 50 μL of cell lysate from cells treated with BoNT / A was mixed with α-SNAP-25 capture antibody solid support and α-GAPDH. Capture antibody solid support was added to each well, the plate was sealed, and the sealed plate was incubated at 4 ° C. for 2-4 hours to overnight on a shaker rotating at 500 rpm. 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Washed twice. After washing, 100 μL of detection antibody solution containing 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) and 1 mg / mL α -SNAP-25 polyclonal antibody / HRP is added to each well of the α-SNAP-25 capture antibody solid support, the plate is sealed and the sealed plate is rt on a shaker rotating at 650 rpm. And incubated for 1 hour. Similarly, 100 μL of detection antibody solution comprising 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) and 0.25 mg / mL α-GAPDH G9545 polyclonal antibody / HRP (Sigma Co., St Louis, MO) is added to each well of the α-GAPDH capture antibody solid support, the plate is sealed, and the sealed plate is incubated at room temperature. Placed on a shaker rotating at 650 rpm for 1 hour at room temperature. After incubating the detection antibody, the wells were washed 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 100 μL of SuperSignal ELISA Pico 1: 1 mixture (Pierce Biotechnology, Inc., Rockford, IL) was added to each well and read at 395 nm using a luminometer (Molecular Devices, Sunnyvale, Calif.). The collected data was analyzed and the EC ₅₀ was calculated as described in Example 5. The results indicate that the data points collected for the amount of detected α-SNAP-25 antibody-antigen complex fit well after being normalized to the amount of detected α-GAPDH antibody-antigen complex. This has been shown to produce more accurate readings. These results indicate that the BoNT / A representative of EC _{50 with} a signal to noise ratio of about 15: 1 to about 20: 1 for the lower asymptote and a signal to noise ratio of about 20: 1 to about 500: 1 for the upper asymptote. The value 1.0 pM was shown to be detected (ranging from about 0.3 pM to about 2.0 pM).

Similar design, the same alpha-GAPDH antibody pair using ECL sandwich ELISA with the BoNT / A cleavage SNAP-25 cleavage product specific for alpha-SNAP having a carboxyl-terminus at the _{P 1} residue of the site scissile bond It can be used in multiplex immune-based methods to detect BoNT / A activity by detecting SNAP-25 cleavage products using -25 monoclonal antibodies.

Immune-based methods for detecting picomolar concentrations of BoNT / A The following examples are illustrative of picomolar concentrations, eg BOTOX®, DYSPORT® / RELOXIN®, PURTOX® Illustrates how to implement an immuno-based method of detecting BoNT / A activity that can detect BoNT / A pharmaceuticals such as XEOMIN®, NEURONOX® or BTX-A.
1. Immune-based method of detecting BoNT / A using an ECL sandwich ELISA To prepare a lysate from cells treated with BoNT / A, approximately 50,000 to 150,000 cells were established from an established cell line. Contains 100 μL of serum-free medium containing 2 mM GlutaMAX ™ I, 1 × B27 supplement, 1 × N2 supplement, 0.1 mM non-essential amino acids, 10 mM HEPES and 25 μg / mL GT1b with minimal essential medium, Earl salt Seed wells of 96-well tissue culture poly-D-lysine plates (see Examples 1 and 2). These cells are differentiated under approximately 5% carbon dioxide in a 37 ° C. incubator until the cells are differentiated as assessed by standard and routine morphological criteria such as growth inhibition and neurite outgrowth (approximately 2 to 3 Day). Medium from differentiated cells was aspirated from each well and reconstituted with sodium chloride-free solution 0 (untreated sample), 0.03 pM, 0.1 pM, 0.3 pM, 0.9 pM, 2.8 pM 8.3 pM or 25 pM BoNT / A pharmaceutical; or 0 (untreated sample) reconstituted with sodium chloride free solution, 0.7 units / mL, 2.1 units / mL, 6.2 units / mL, Replaced with fresh medium containing either 18.5 units / mL, 55.6 units / mL, 166.7 units / mL or 500 units / mL BoNT / A drug. Since BoNT / A pharmaceutical contains sodium chloride, its addition to the culture medium resulted in a hypertonic medium that is detrimental to cell viability. To avoid the hypertonic problem, 200 μL of MEM medium made without sodium chloride was used to reconstitute the BoNT / A drug product to obtain a final concentration of 25 pM BoNT / A (500 units / mL). . A dose-response curve for the total concentration by adding sodium chloride to the medium used to make the dilution correspond to the amount of excipient present at the highest concentration used (25 pM or 500 units / mL). The matrix was kept constant along After 24 hours of treatment, cells were washed and incubated for an additional 2 days without toxin. To harvest the cells, the medium is aspirated, washed with 1 × PBS, and a lysis buffer comprising 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl ₂ , 1 mM EGTA, 1% Triton X-100 is added to each well. The plate was lysed by addition and the plate was incubated for 30 minutes at 4 ° C. on a shaker rotating at 500 rpm. The plate was centrifuged at 4000 rpm for 20 minutes at 4 ° C. to pellet cell debris and the supernatant was transferred to a capture antibody coated 96 well plate to perform the detection step.

  A solid support comprising an α-SNAP-25 capture antibody solution, an α-SNAP-25 detection antibody solution, and a capture antibody specific for the SNAP-25 cleavage product, as described in Example 6. Prepared.

To detect the presence of SNAP-25 cleavage product by ECL sandwich ELISA analysis, aspirate blocking buffer from the stored plate, add 25 μL of lysate from cells treated with BoNT / A, and Plates were incubated at 4 ° C. for either 2 hours or 24 hours. 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Washed twice. After washing, 5 μg / mL α-SNAP-2 detection comprising 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) 25 μL of antibody solution was added to each well, the plate was sealed and the sealed plate was incubated with shaking for 1 hour at room temperature. After incubating the α-SNAP-25 detection antibody, the wells were washed 3 times with 200 μL of 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, the plate was processed, the collected data was analyzed, and the EC ₅₀ was calculated as described in Example 5. These results indicate that the BoNT / A representative of EC _{50 with} a signal to noise ratio of about 15: 1 to about 20: 1 for the lower asymptote and a signal to noise ratio of about 20: 1 to about 500: 1 for the upper asymptote. A value of 1.0 pM was detected (range from about 0.3 pM to about 2.0 pM) (FIG. 9). This method can also be performed in a multiplex fashion as described in Example 8.

2. Immune-based method of detecting BoNT / A using CL sandwich ELISA A lysate and α-SNAP-25 capture antibody solution from cells treated with BoNT / A is prepared as described in Example 6. . A solid support comprising an α-SNAP-25 detection antibody solution and a capture antibody that is specific for the SNAP-25 cleavage product is prepared as described in Example 7.

To detect the presence of SNAP-25 cleavage product by CL sandwich ELISA analysis, 25 μL of lysate from cells treated with BoNT / A was added to each well, the plate was sealed, and the sealed plate was placed at 500 rpm. Incubated at 4 ° C for either 2 or 24 hours on a shaker rotating at 3 plate wells by aspirating the cell lysate and rinsing each well 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) Wash once. After washing, 1 mg / mL α-SNAP-25 polyclonal comprising 2% Amersham blocking reagent in 1 × PBS, 0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate) 100 μL of antibody / HRP detection antibody solution was added to each well, the plates were sealed and the sealed plates were incubated for 1 hour at room temperature on a shaker rotating at 650 rpm. After incubating the detection antibody, the wells are washed 3 times with 200 μL of 1 × PBS, 0.05% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). After washing, 100 μL of SuperSignal ELISA Pico 1: 1 mixture (Pierce Biotechnology, Inc., Rockford, IL) is added to each well and read at 395 nm using a luminometer (Molecular Devices, Sunnyvale, Calif.). The collected data is analyzed and an EC ₅₀ is calculated as described in Example 5. This method can also be performed in a multiplex fashion as described in Example 8.

Immune-based methods for detecting α-BoNT / A neutralizing antibodies The following examples illustrate how to implement immuno-based methods that can detect the presence of α-BoNT / A neutralizing antibodies.
BoNT / A is currently used in a wide range of medical applications, including muscle hyperactivity, ophthalmology, gastrointestinal tract, urology and cosmetics. Repeated long-term treatment with BoNT / A can cause patients to develop α-BoNT / A neutralizing antibodies against the toxin, leading to immune resistance. The α-BoNT / A neutralizing antibody binds to the binding domain (H _C ) and / or the translocation domain (H _N ) of BoNT / A, thereby stopping the uptake of toxins into neuronal cells. Block activity. Several studies suggest that up to 5-10% of patients treated repeatedly with BoNT / A prescriptions for dystonia have immune resistance by developing α-BoNT / A neutralizing antibodies. Yes. An established assay for determining the presence of α-BoNT / A neutralizing antibodies in a patient's blood is the mouse protection assay (MPA). Currently, BoNT / A is incubated with patient serum prior to injection into mice. The presence of the antibody is manifested by a reduced response to the neurotoxin in the animal. Since MPA is an in vivo based assay, it will be more cost and time efficient if it is replaced by a cell based assay.

  To detect the presence or absence of α-BoNT / A neutralizing antibodies, the immuno-based methods for determining BoNT / A activity disclosed herein can be used. One format uses Western blot detection to determine the amount of SNAP-25 cleavage product present after treatment with various concentrations of BoNT / A, and another format is ECL sandwich ELISA detection. Was to use the method.

  Serum was isolated from the blood of different individuals to prepare a sample comprising an α-BoNT / A neutralizing antibody and a negative control sample known to lack α-BoNT / A neutralizing antibody. . Individuals with attenuated immunity were referred to as naive individuals. Individuals who acquired immunity were referred to as immunized individuals. Blood was placed in a serum tube containing a procoagulant (BD Biosciences, Bedford, MA). Serum was obtained by centrifuging blood at 1,000 × g for 10 minutes at 4 ° C. Sera from two donors were obtained: one solid was immunized against BoNT / A, but the other was not.

  To prepare lysates from cells treated with a sample comprising BoNT / A, SiMa cells were seeded into poly-D-lysine 96-well plates and differentiated as described in Example 6. Human serum was serially diluted 1: 100-1: 152,000 using a serum-free medium in a 2.5-fold increase. BoNT / A was suspended at a concentration of 10 pM in 0.5 mL of SiMa culture medium. Medium containing BoNT / A and α-BoNT / A antibody was mixed and incubated at room temperature for 15 minutes or 1 hour. Cells were treated with BoNT / A with human serum for 2 hours followed by incubation in fresh growth medium for 15 hours. Cells were also treated for 15 hours without further incubation time.

To detect the presence of SNAP-25 cleavage product by Western blot analysis, media was aspirated from each well, cells were suspended in 50 μL of SDS-PAGE loading buffer, and then heated to 95 ° C. for 5 minutes. Samples collected using a 12% 26 well Criterion gel (Bio-Rad Laboratories, Hercules, CA) were separated from each collected sample as described in Example 1 except that the samples were separated by SDS-PAGE. Aliquots were analyzed by Western blot and rabbit polyclonal α-SNAP-25 ₁₉₇ antibody serum was used as the primary antibody (see Example 4). The results show that the test sample resulted in reduced cleavage of SNAP25 when compared to the negative control sample, thereby demonstrating that serum from the immunized individual contained α-BoNT / A neutralizing antibody. Is done.

To detect the presence of SNAP-25 cleavage product by ECL sandwich ELISA, media was removed from each well and cells were lysed as described in Example 5. α-SNAP-25 capture antibody solution, α-SNAP-25 detection antibody solution and α-SNAP-25 solid support were prepared as described in Example 7. The supernatant was transferred to an α-SNAP-25 solid support and an ECL sandwich ELISA assay was performed as detailed in Example 5. The collected data were analyzed and the SNAP-25 cleavage product obtained at the highest dilution of serum, with the EC ₅₀ being the serum dilution required to block BoNT / A activity to half of its maximum By dividing the product signal by the signal obtained at the lowest serum dilution, a ratio of maximum signal (signal _Max ) to minimum signal (signal _Min ) was obtained, as described in Example 5 with an EC ₅₀ Calculated.

  The results indicate that the presence of α-BoNT / A neutralization in human serum can be detected. The activity of the BoNT / A complex incubated in serum from immunized individuals decreased with decreasing serum dilution, while the presence of naive serum was not affected at any dilution tested. There wasn't. This assay can be performed using formulated BoNT / A pharmaceuticals, bulk BoNT / A complexes or purified neurotoxins.

Immune-based methods for detecting BoNT / A activity using engineered cells The following example introduces a polynucleotide molecule encoding a BoNT / A receptor into cells from an established cell line to form BoNT Explains how to further improve susceptibility to / A addiction or improve BoNT / A uptake capacity.
A polynucleotide molecule of SEQ ID NO: 130 that encodes FGFR2 of SEQ ID NO: 59, or SEQ ID NO: by the cationic lipid method for introducing an exogenous BoNT / A receptor into a cell comprising an established cell line An expression construct comprising the polynucleotide molecule of SEQ ID NO: 139 encoding 25: FGFR3 was transfected into cells from an established cell line. Cells from an established cell line of appropriate density (approximately 5 × 10 ⁶ cells) are plated on 100 mm tissue culture dishes containing 5 mL of complete culture medium and cells are grown under 5% carbon dioxide in a 37 ° C. incubator. Grow until the appropriate density for transfection is reached. 1.5 mL of OPTI-MEM reduced serum medium containing 60 μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) Incubated for 5 minutes at room temperature, 24 μg of expression construct encoding FGFR2 or FGFR3, or green fluorescent protein (GFP). Prepare 3 mL of transfection solution by adding to 1.5 mL of OPTI-MEM reduced serum medium containing the encoded control expression construct. The transfection mixture was incubated for approximately 30 minutes at room temperature. The complete medium is replaced with 3 mL of transfection solution and the cells are incubated for approximately 8 hours under 5% carbon dioxide in a 37 ° C. incubator. The transfection medium is replaced with 3 mL of fresh complete medium and the cells are incubated for approximately 24 hours under 5% carbon dioxide in a 37 ° C. incubator. The medium is replaced with 3 mL of fresh complete medium containing approximately 1 mM G418 (Invitrogen, Carlsbad, CA). Cells are incubated for approximately 1 week under 5% carbon dioxide in a 37 ° C. incubator and the old medium is replaced with fresh complete culture medium containing 0.5 mM G418. Once an antibiotic resistant colony is established, the resistant colony is placed in a new 100 mm culture plate containing fresh complete culture medium supplemented with approximately 0.5 mM G418 and the cells are transferred to 6-20 × 10 ⁵ cells / cell. Swell again until a density of mL is reached.

  Treatment with various doses of BoNT / A complex to determine whether BoNT / A receptor overexpression improved cell sensitivity to BoNT / A poisoning or improved BoNT / A uptake capacity Dose-response curves were generated using the prepared cells. To prepare lysates from cells treated with BoNT / A, the appropriate density of cells from an established transfected cell line was added to 96 wells containing 100 μL of appropriate serum-free medium (Table 5). Seeded into wells of tissue culture plate. These cells are differentiated (approximately 3 days) under 5% carbon dioxide in an incubator at 37 ° C. as assessed by standard and routine morphological criteria such as growth arrest and neurite outgrowth. Incubated. Medium from differentiated cells is aspirated from each well and for cells comprising SiMa or PC12 transfected cell lines 0 (untreated sample), 0.01 nM, 0.04 nM, 0.12 nM, 0 For cells comprising .37 nM, 1.1 nM, 3.3 nM and 10 nM BoNT / A complex; and Neuro-2a transfected cell lines, 0 (untreated sample), 0.14 nM, 0.40 nM, It was replaced with fresh medium containing either 1.2 nM, 3.7 nM, 11 nM, 33 nM and 100 nM BoNT / A complex. Cells are treated with BoNT / A containing medium for 6 hours, followed by incubation with fresh medium for 15 hours, and 40 μl of 2 × SDS-PAGE loading buffer is added and the plate is heated to 95 ° C. for 5 minutes. Collected.

Example 1 except that samples collected using a 12% 26 well Criterion gel (Bio-Rad Laboratories, Hercules, CA) were separated by SDS-PAGE to detect the presence of SNAP-25 cleavage products. Aliquots from each collected sample were analyzed by Western blot as described in and using the following primary antibodies: a 1: 1,000 dilution of rabbit polyclonal α-SNAP-25 antibody serum (Examples). 4) (AGN, polyclonal antibody), 1: 500 dilution of α-FGFR2 rabbit polyclonal C-17 (Santa Cruz Biotechnology, Santa Cruz, CA) or 1: 500 dilution of α-FGFR3 rabbit polyclonal C-15 (Santa Cruz Biotechnology) , Santa Cruz, CA). The intensity of the protein of interest from each sample was calculated using Image Quant (GE Healthcare, Piscataway, NJ) and the EC ₅₀ for each of the cell lines was estimated using SigmaPlot software.

The results indicate that cells transfected with FGFR2 or FGFR3 were more sensitive to BoNT / A than cells transfected with GFP and also showed high levels of SNAP-25 cleavage. (Table 14). The EC ₅₀ value for cells overexpressing FGFR2 or FGFR3 is lower than the EC ₅₀ value exhibited by cells overexpressing GFP, indicating that introduction of FGFR2 or FGFR3 improved cell sensitivity to BoNT / A poisoning, It also shows that BoNT / A uptake capacity has been improved.

ＳＮＡＰ−２５切断生成物の存在に関する検出を実施例６−１０に記載されるようにサンドイッチＥＬＩＳＡを用いて実施することもできる。

Detection for the presence of SNAP-25 cleavage product can also be performed using a sandwich ELISA as described in Examples 6-10.

Claims (15)

  A composition comprising a carrier, a mobile linker comprising at least 3 amino acids, and a SNAP-25 antigen comprising SEQ ID NO: 148.   The composition of claim 2, wherein the mobile linker and the SNAP-25 antigen amino acid sequence is SEQ ID NO: 38. The isolated α-SNAP-25 antibody binds to an epitope comprising a carboxyl terminal glutamine from the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product;
The α-SNAP-25 antibody has an association rate constant of less than 1 × 10 ¹ M ⁻¹ s ^{−1 for an} epitope that does not contain the carboxyl terminal glutamine of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product. And α-SNAP-25 antibody has an equilibrium dissociation constant of less than 0.450 nM for that epitope;
Isolated α-SNAP-25 antibody.   The isolated α-SNAP-25 antibody of claim 3, wherein the epitope is SEQ ID NO: 32. a) heavy chain variable region comprising SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80 or SEQ ID NO: 82; and b) SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: A light chain variable region comprising: SEQ ID NO: 90 or SEQ ID NO: 92;
An isolated α-SNAP-25 antibody comprising. a) heavy chain variable region comprising SEQ ID NO: 93, SEQ ID NO: 121 or SEQ ID NO: 100; and b) light chain variable region comprising SEQ ID NO: 105, SEQ ID NO: 110 or SEQ ID NO: 115. ;
An isolated α-SNAP-25 antibody comprising. At least SEQ ID NO: 100 of the _V H CDR3, SEQ ID NO: 101 _V H CDR3 or SEQ ID NO: 102 An isolated α-SNAP-25 antibody comprising a _V H CDR3 of. a) treating cells from an established cell line with a sample comprising BoNT / A, wherein cells from the established cell line are susceptible to BoNT / A poisoning by BoNT / A of about 500 pM or less. high;
b) isolating a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl terminal glutamine with a BoNT / A cleavage site cleavable linkage from the treated cells;
c) contacting the SNAP-25 component with an α-SNAP-25 antibody linked to a solid support;
Wherein α-SNAP-25 antibody binds to an epitope comprising the carboxyl terminal glutamine of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product, and α-SNAP-25 antibody produces SNAP-25 cleavage. BoNT / A cleavage site from the product has an association rate constant of less than 1 × 10 ¹ M ⁻¹ s ^{−1 for} an epitope from SNAP-25 that does not contain the carboxyl terminal glutamine of the cleavable bond; and α-SNAP-25 The antibody has an equilibrium dissociation constant of less than 0.450 nM for the epitope;
d) detecting the presence of an α-SNAP-25 antibody and an antibody-antigen complex comprising a SNAP-25 cleavage product having a carboxyl-terminal glutamine from the BoNT / A cleavage site cleavable linkage;
Here, detection by antibody-antigen complex is an indicator of BoNT / A activity;
A method for detecting BoNT / A activity comprising the steps of: 9. The method of claim 8, wherein the SNAP-25 cleavage product is SNAP-25 ₁₉₇ .   9. The method of claim 8, wherein the presence of the antibody-antigen complex is detected using a sandwich ELISA.   9. The method of claim 8, wherein the method has a signal to noise ratio of at least 3: 1 at the lower asymptote and a signal to noise ratio of at least 10: 1 at the upper asymptote.   9. The method of claim 8, wherein the sample comprises at most 100 pM BoNT / A.   9. The method of claim 8, wherein cells from the established cell line are sensitive to BoNT / A intoxication with about 100 pM or less of BoNT / A.   9. The method of claim 8, wherein the method is performed in a single plex mode or a multiplex mode. a) adding BoNT / A to a test sample obtained from a mammal under test for the presence or absence of α-BoNT / A neutralizing antibodies;
b) treating cells from an established cell line with a test sample, wherein cells from the established cell line are sensitive to BoNT / A poisoning;
c) isolating a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminal glutamine from a BoNT / A cleavage site cleavable linkage from the treated cells;
d) contacting the SNAP-25 component with an α-SNAP-25 antibody linked to a solid support, wherein the α-SNAP-25 antibody is;
Wherein α-SNAP-25 antibody binds to an epitope comprising the carboxyl terminal glutamine of the BoNT / A cleavage site cleavable linkage from the SNAP-25 cleavage product, and α-SNAP-25 antibody produces SNAP-25 cleavage. BoNT / A cleavage site from the product has an association rate constant of less than 1 × 10 ¹ M ⁻¹ s ^{−1 for} an epitope from SNAP-25 that does not contain the carboxyl terminal glutamine of the cleavable bond; and α-SNAP-25 The antibody has an equilibrium dissociation constant of less than 0.450 nM for that epitope;
e) detecting the presence of an α-SNAP-25 antibody and an antibody-antigen complex comprising a SNAP-25 cleavage product having a carboxyl-terminal glutamine from a BoNT / A cleavage site cleavable linkage;
f) repeating step be with a negative control sample instead of a test sample, the negative control sample comprising BoNT / A and serum known to contain no α-BoNT / A neutralizing antibody; And g) comparing the amount of antibody-antigen complex detected in step e with the amount of antibody-antigen complex detected in step f;
Here, the smaller amount of antibody-antigen complex detected in step e relative to the amount of antibody-antigen complex detected in step f is an indicator of the presence of α-BoNT / A neutralizing antibody. ;
A method for determining BoNT / A immunoresistance in a mammal comprising the steps of:

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